R. Verbeke

Senstitive multi-residue method for detection of anabolics in urine and in tissues of slaughtered animals

A routine procedure is described for the dependable detection of various anabolic residues in tissues or urine contaminated at levels as low as 0.5–10ppb (10 parts per 109). A suitable extraction and clean-up procedure was developed. permitting adequate recovery (60–80%) of various anabolics from tissue samples (50 g) or urine (50 ml). Follwong two-dimensional thin-layer chromatography, the presence of the anabolic residues are detected by sulphuric acid—induced fluorescence. The detection limit of most anabolics is of the order 1–10 ng.

Detection of antithyroid residues in meat and some organs of slaughtered animals

A simple method is described for the routine detection of antithyroid residues in thyroid, liver, kidney and meat contaminated at levels as low as 10 ppb (10 parts per 10(9)). Tissue samples (2 g) are homogenized in methanol, contaminating lipids and amino acids are removed and the antithyroid residues are subjected to reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in buffer. The NBD derivatives are extracted with diethyl ether and separated by thin-layer chromatography. After spraying with cysteine or mercaptoethylamine, the antithyroid residues appear as fluorescent spots. The detection limit of these compounds is of the order of 200 pg.

Determination of thiocyanate in tissues and body fluids of animals by gas chromatography with electron-capture detection

A method for the quantitative extraction of thiocyanate from biological material has been developed. Levels of 0.2-5 ppm of thiocyanate, in the presence of cyanide, were determined by gas-solid chromatography using 2-bromopropane as internal standard. Reliable results can be obtained only after careful deproteinization of the extracts with hot methanol. Cyanide can be eliminated by reaction with alkaline formaldehyde. The reproducibility of the determination of thiocyanate in biological extracts was +/-5%; the recoveries were over 90%. In vivo experiments with KS14CN-treated rats gave good agreement between the analytical results and the amount of thiocyanate determined by isotope dilution.

Quantitative determination of androstenone in pig adipose tissue

A simple, cheap and rapid method for the quantitative determination of the boar taint substance, 5 alpha-androst-16-en-3-one, in pig adipose tissue is described. After saponification of the fat the androstenone is extracted, derivatised with o-(pentafluorobenzyl)hydroxylamine hydrochloride in pyridine and analysed by fused-silica open-tubular capillary gas chromatography with electron-capture detection.

Sensitive and specific post-column fluorimetric determination of diethylstilboestrol residues in extracts of urine and animal tissues at the 1-ppb level

A rapid method for the selective detection and quantitation of diethylstilboestrol (DES) residues at the 1 ppb level in extracts of urine and animal tissues is described. After selective extraction of the oestrogens, DES is analysed by high-performance liquid chromatography using an in-line specific photochemical reactor followed by oxidation to highly fluorescent products. The reaction products of DES were investigated by thin-layer chromatography (TLC). The specificity of the proposed method was compared with that of a reference TLC method on different extracts of animal origin.

Variations in the concentrations of free amino acids in the plasma of the dairy cow at parturition

The plasma levels of individual amino acids were studied in 6 dairy cows from 4 days before to 3 days after calving. During this sampling period, the concentrations of 13 amino acids showed significant changes. The levels of several amino acids were depressed markedly in the sample collected immediately before calving. Following parturition, the concentration of most amino acids gradually returned to values obtained 3 days before calving. The glutamine and alanine contents of the plasma rose to a peak value 1 day after calving and subsequently decreased. The mean concentrations of glycine and α-aminobutyric acid did not change before parturition but rose significantly thereafter. These observations are discussed in terms of amino-acid utilization for milk protein synthesis and gluconeogenesis at the onset of lactation. The changes in plasma amino acid levels appear to be synchronized with those reported for prolactin and progesterone in the 24 h before parturition. This may indicate an important influence of both hormones on the lactogenic process in the cow. The highly significant correlations obtained between the concentrations of 14 individual amino acids are discussed.

Metabolism of [U-14C; 2, 3-3H]-L-valine by the isolated perfused goat udder

Two lactating mammary glands excised from 2 goats were perfused for several hours in the presence of [U-14C; 2,3-3H]-L-valine and received adequate quantities of glucose, acetate and amino acids. In the synthesized milk 96 and 89% respectively of the casein valine was derived from free plasma valine. Valine was extensively catabolized by mammary tissue, resulting in a considerable 14CO2 production and in the incorporation of 14C into milk citric acid and to a lesser extent into casein aspartic acid and glutamic acid. About 30% of the valine molecules which were taken up by the mammary gland were oxidized to CO2 and 70% were incorporated in casein as valine residues. About 10% of the plasma valine molecules were reversibly transaminated during one passage through the udder. An important amount of radioactivity of plasma was present in unknown metabolites. Only 7% of this activity was localized in isobutyrate. The radioactivity of total milk fat was very low. Mainly iso-14:0, iso-16:0 and 15:0 were labelled.

Analysis of anti-hormones

Abstract Residues of anti-hormones in biological materials are routinely determined at the ppb-level 1 by specific detection through high-performance thin-layer chromatography with fluorimetric detection or capillary gas chromatography with electron-capture detection backed up by a selective single step extraction.

Incorporation of 1-14C and [3-14C]-butyrate into milk constituents by the perfused cow's udder.

One half-udder from a lactating cow was perfused for 110 min with blood containing 1 mc of [1- 14 C]butyrate. Inactive sodium butyrate was added at the eightieth minute. The volume of milk collected was 320 ml. One half-udder from another cow was perfused for 120 min in the presence of 0·5 mc [3- 14 C]butyrate, inactive sodium acetate being added at the sixtieth minute. The volume of milk collected was 150 ml. The total amount of 14 CO 2 recovered was 5·7% of the [1- 14 C]butyrate given and 7·6% of the [3- 14 C]butyrate given. In both instances casein, followed by the volatile fatty acids, showed the highest specific activity of the constituents isolated from the milk. The glyceride fatty acids in the udder were fifty times as active as those in the milk. With [3- 14 C]butyrate the activity of the total fatty acids amounted to 24% and that of total lactose to 0·38% of the added 14 C. Butyrate did not appear to be used for glycogenesis in the perfused gland. The specific activity of the lower fatty acids of both the udder and the milk increased stepwise with increasing chain length to reach a maximum at C 10 . A satisfactory explanation for this peculiar 14 C distribution cannot be given at the present time. There was no evidence of direct esterification of butyrate. Most of the activity of casein was due to labelled glutamic and aspartic acids, the activity of the former being four times as high as that of the latter. The acids of the Krebs cycle isolated from the udder tissue when [3- 14 C]butyrate was given showed very high activity. No striking differences were observed between the results of the two experiments. It is concluded that butyrate is split into two C 2 components which behave identically. These are utilized for fatty acid synthesis and take part in the Krebs cycle. The relative 14 C distribution between the components isolated from milk and those from tissue may be a reflexion of the secretory processes in the udder cells, synthesized fat tending to be secreted in the alveoli after the other constituents.

Determination of oxazolidine-2-thiones in biological fluids in the ppb range

Abstract A simple, cheap and rapid method for the quantitation of oxazolidine-2-thiones in biological fluids and fodder in the concentration range 1–1000 ppb is described. Oxazolidine-2-thiones are extracted from 2 ml fluid using cyclohexane saturated with phenylmercuric acetate, derivatized with pentafluorobenzoyl chloride and analysed by fused-silica open tubular capillary gas chromatography with electroncapture detection.