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A flow injection manifold is described based on a flow-through arrangement of thermistors for measuring heat effects of chemical reactions. Temperature changes down to 10 °C can be measured reproducibly. The working range for the determination of a component is dependent on the reaction enthalpy and can vary from 10 to 10 mol/l for acids and bases, from 10 to 1000 ppm for e.g., alkylhydroperoxides. Sample frequencies up to 150 samples per hour can be realized. [ABSTRACT FROM AUTHOR]

Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ss-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC-MS/MS. The method was validated at the current MRLs, which range from 25-300 microg kg(-1) in muscle and kidney to 4-30 microg kg(-1) in milk as well as at the target value of 100 microg kg(-1) chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94-113% (muscle), 83-111% (kidney) and 87-103% (milk) to 88-116% (animal feed). Intra-day precision (relative standard deviation (RSD)(r)) ranged from 5-13% (muscle, n = 18), 4-17% (kidney, n = 7) and 5-18% (milk, n = 7) to 11-32% (animal feed, n = 18). Inter-day precision (RSD(RL), n = 18) ranged from 6-23% (muscle) to 11-36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed.

A bioassay was developed for the detection of a broad range of beta-agonist compounds in animal feeds. A solubilised beta2-adrenoceptor was utilised as the binding protein in the assay. This protein was found to be highly stable when stored at 80 degrees C. The assay was developed and initially validated to determine the sensitivity and relative selectivity against a panel of commonly used beta-agonist compounds. It was also shown that when beta-agonists were present as cocktails in samples a pronounced synergistic effect could be measured. The method was further validated according to EC Decision 2002/657 and proved capable of detecting 250ng clenbuterol equivalents per gram of sample. This is well below the quantities normally associated with beta-agonist medicated feeds. The beta2-adrenoceptor used in the study only failed to bind the compound zilpaterol, raising doubts as to whether this compound is a true beta2-adrenergic drug.

The use of nitrofurans as veterinary drugs has been banned in the EU since 1993 due to doubts on the safety of the protein-bound residues of these drugs in edible products. Following treatment of pigs with the veterinary drug furazolidone free 3-amino-2-oxazolidinone (AOZ), the side-chain of the drug, could be detected in the blood in concentrations up to 0.3 μg/ml. The identity of the free AOZ was confirmed by LC/MS. This shows that the side-chain can be released from the parent drug, most likely under the acidic conditions in the stomach. Free AOZ was also detected in the blood of rats fed pig liver with protein-bound residues of furazolidone. Incubation of isolated pig hepatocytes with radiolabeled AOZ, resulted in the formation of protein-bound metabolites, to a similar extent as observed with furazolidone itself. Much lower levels were formed in the presence of dimethylsulfoxide or 4-chlorobenzenesulfonamide, most likely due to inhibition of the enzyme involved in the metabolic activation of AOZ. These compounds also prevented the inhibition by AOZ of monoamine-oxidase (MAO) activity in pig hepatocytes. These data strongly indicate that the protein-bound metabolites of furazolidone in tissues of treated animals are derived following metabolic activation of furazolidone itself, but also of the free AOZ side-chain, following its release from the parent drug. In addition to the MAO-inhibition and formation of protein-adducts, AOZ gave a dose-related positive respons in the Salmonella/microsome mutagenicity test especially in the presence of rat liver S9-mix, in tester strains TA 1535 and TA 100. Furthermore, a positive response was obtained in the chromosome aberration test with human lymphocytes and in the bone marrow micronucleus test with mice treated intraperitoneally with AOZ. It is concluded that ingestion of protein-bound residues of furazolidone results in the release and absorption of AOZ, a compound with potential mutagenic properties. This is the first report showing that protein-bound residues of veterinary drugs can be of toxicological significance.

Microcolumn liquid chromatography (microLC) combined with electrospray tandem mass spectrometry is used for the determination of intact glycosylated cytokinins and the corresponding aglycons at picomole and sub-picomole levels in plant tissue. Routine analysis was done on C8-bonded silica using a methanol-water gradient. Data acquisition was performed by multiple reaction monitoring. Quantification was carried out by using isotopically labelled analogues and applying linear regression to the response factor versus concentration data. For routine analysis a calibration range from 0.5 to 10 pmole injected on-column was used. The limits of detection ranged from 50 to 100 fmole injected on-column. The microLC procedure was used to analyse plant tissue extracts from transgenic homozygote and hemizygote as well as wild-type Nicotiana tabacum species, and cauliflower samples. The data were compared with results obtained by conventional immunoassay and a satisfactory correlation was found. Validation data are presented.

A method is described for the analysis of beta-agonists in urine of cattle. The method uses solid-phase extraction (SPE), followed by analysis of the resulting extract by flow injection thermospray tandem mass spectrometry (TSP-MS-MS). Sample preparation is performed using a mixed-bed SPE procedure using a sorbent having both hydrophobic and ionic properties. MS-MS analysis following thermospray ionization, is performed in single-reaction monitoring parent mode. In that way isotope dilution can be used for quantitation of clenbuterol. Data are presented on precision and accuracy for clenbuterol and related compounds. Furthermore, data acquisition was performed in full-scan neutral loss mode to indicate the suitability of flow injection analysis (FIA)-TSP-MS-MS for exploratory analysis. Detection of beta-agonists in this mode is based on the presence of the N-tert.-butyl-beta-ethanolamino moiety and, in that respect, detection of known as well as unknown compounds having this moiety will take place. This feature is exemplified by the analysis of samples containing several compounds.

A previously described method for the confirmatory analysis of clenbuterol with chemical ionization GC-MS was extended to fit more beta-agonists. The method has been used routinely for about one year and a large number of samples have been analysed according to the described procedure. This paper summarizes the results obtained with this method with regard to clenbuterol and furthermore discussion is focused on applicability to beta-agonists other than clenbuterol with respect to the European Community requirements for confirmatory analysis.

To meet the requirements of the European Community for confirmatory analysis of clenbuterol using low-resolution mass spectrometry, usually two different techniques (i.e. electron impact and chemical ionization) have to be applied to confirm unambiguously its presence in extracts of urine. This paper describes the application of two different derivatives and the simultaneous analysis of these two different derivatives in one gas chromatographic-mass spectrometric analysis. With the proposed combination of techniques, Community requirements can more easily be met in only one analytical run. Examples of the analysis of some urine samples are presented, as well as data on linearity, repeatability and equivalence of the combined technique to separate determinations.