Comparison of purification procedures for the isolation and detection of anabolic residues in faeces using gas chromatography-mass spectrometry.

Within several regional field laboratories and the national reference laboratory a harmonised methodology for the analysis of anabolic residues in faecal samples was developed. The method consists of a liquid-liquid and a solid-phase extraction step, followed by a high-performance liquid chromatography purification step. Using gas chromatography-mass spectrometry, currently illegally used anabolic steroids can be detected in faeces at the ppb level. Within this context acidification, followed by centrifugation under cooling, allows efficient, practical and rapid defatting of faecal samples. Furthermore, a combination of a silica and an aminopropyl solid-phase extraction column was found to give the best results as regards the sample purification process.

Can measurements of potential doubling time (Tpot) be compared between laboratories? A quality control study.

The purpose of this study was to investigate the reproducibility of potential doubling time measurements of human tumors in different laboratories and to distinguish which steps in the measurement procedure were subject to the greatest variation. This was achieved by comparing measurements on the same source material in two different laboratories in which three aspects of the technique were separately studied, namely, preparation and staining of the nuclear suspensions, running the samples on the flow cytometer (FCM), and analyzing the two-parameter FCM data. This involved exchange between the two centers of fixed tumor material, stained nuclear suspensions, and FCM data on floppy disks. The analysis step was found to be the least variable step. For DNA synthesis time, Ts, and the labeling index, LI, the coefficients of determination (R2) ranged from 92% to 95.4%. A small systematic bias was observed, with one center measuring approximately 15% higher values for both LI and Ts. Different criteria for window placements were found to be a contributing factor. Variations in the FCM step were approximately equal to those for analysis, with no systematic deviations. Variations for the preparation and staining step were the largest (R2 = 60.5% and 38.1% for LI and Ts, respectively). However, this step was the only one subject to intratumoral variability, which was the largest contributing factor to the variations observed. In addition, however, LI was on average 41% higher in one center compared to the other, resulting in a systematic bias. Differences in the level of green fluorescence of the labeled cells implicated antibody differences as a possible cause. The variations found here for the three procedural aspects were significantly smaller than variations observed between tumors, a requirement for a predictive assay. Suggestions for implementation of quality control procedures include objective (computer-assisted) data analysis on two-parameter histograms and optimization of antibody combinations.

By-products of steroid synthesis: a cause of interferences in thin-layer chromatography residue analysis.

Since the late 1980s all of the laboratories involved in high-performance thin-layer chromatography (HPTLC) control of hormonal residues in kidney fat, have occasionally detect a green fluorescent spot with similar RF values and colour to those observed for methyltestosterone (MT). This spot (product) could lead to false positive results for MT and was thus named 'le faux méthyl' (the false methyl) by a french speaking colleague. All of the samples with a false methyl spot also contained a relatively high concentration of progesterone. Differentiation of this product from methyltestosterone can be performed in three ways: firstly, extra HPTLC on reversed-phased plates, secondly, extra purification of the extract with HPLC prior to HPTLC and thirdly, gas chromatography--mass spectrometry. This interference was identified as 20 beta-hydroxyprogesterone, a by-product of progesterone. The problem of the false methyl was not only linked with the TLC characteristics of MT but also to the progesterone used as standard. Some laboratories used an analytical-reagent grade standard and others used commercial progesterone powders as standards (e.g., obtained in crude form from pharmaceutical companies). The commercial-grade progesterones showed two spots in comparison with the analytical standard that showed just one spot. As the false methyl was observed not only in kidney fat and meat samples, but also in illegal hormone cocktails, it was concluded that we had detected a by-product of an illegally used 'natural progesterone'.

Quality of the meat after the application of anabolic agents in young calves.

Experiments were carried out to test the effect of implantation of Implix¿ (20 mg estradiol + 200 mg testosterone) or Revalor¿ (140 mg trienbolone acetate + 20 mg estradiol) on growth, feed conversion, slaughter quality, and residue levels in black Friesian bull calves at 4, 8, or 4 and 8 weeks before slaughtering. Weighings and calculations of feed conversion were carried out weekly. The control group of weight gain and feed conversion consisted of 22 calves; the chemical analyses of the control group was restricted to 8 animals. Implantation of the hormones at 4 or 8 weeks before slaughtering resulted in a significant beneficial effect on weight gain, feed conversion and carcass index over the control. This effect was improved (up to 15%) by implanting the hormones at 8 and 4 weeks before slaughter. The effects obtained with Revalor seemed to be superior to those obtained with Implix. In 75% of the treated animals, some remainder of implants was found. No effect was found on water binding capacity and colour of raw meat. Carcass quality was not significantly improved as judged from water, fat, ash, and protein content of the M. Longissimus Dorsi. The collagen content of the animals treated with Revalor was slightly, though not significantly, increased. There is a slight decrease in the relative bone content of the treated animals over the control resulting in higher meat percentages. Residues were determined in meat obtained from rib and neck by biological and chemical methods. Oestrogenic activity was found in only 7 samples from the neck; all samples from the rib were negative. Chemical examination indicates the presence of oestradiol in these samples. No residues of trienbolone could be detected in the meat samples. The Pars Dissiminata of the prostate was examined histologically on frozen and paraffin sections. Although the latter permitted a sharper interpretation, both methods indicated an increased activity of the prostates induced by hormone treatment. Compared to Implix, Revalor treatment provoked a more pronounced mucous activity. In recent years, the administration of estrogens in combination with testosterone or with trenbolone acetate (androst-4,9[10]-11-trien-3-one 17-acetate)2,3,4,17 has been shown to improve the growth rate and feed conversion5,14,17 in farm animals. However, there is a lack of information on carcass quality, which is of interest to the producer as well as to the consumer. Moreover, there are sample data14 about the residue levels remaining in the carcass following hormone implantation. These experiments were carried out to test the effect of the implantation of Implix¿ (20 mg estradiol + 200 mg testosterone) or Revalor¿ (140 mg trienbolone acetate + 20 mg estradiol) on growth, feed conversion, slaughter quality, and residue levels in black Friesian bull calves.