17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle.

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.

Metabolites in feces can be important markers for the abuse of anabolic steroids in cattle.

In Belgium, to control the abuse of anabolic steroids in cattle, urine samples have been gradually replaced by feces samples, because the latter can be obtained more easily from living animals. Urine and feces samples were collected from heifers after administration of boldenone, norethandrolone or ethylestrenol. Metabolites present in feces or urine were determined by GC-MS. Large qualitative and quantitative differences in the metabolic profiles were observed. In feces, in contrast to urine, the parent compounds or their major metabolites were detectable only shortly after administration. On the other hand, metabolites resulting from the reduction of the 3-oxo group and the unsaturated carbon-carbon bonds, present on the A-ring, allow for long-term detection in feces. A-ring reduced metabolites have been identified in samples found positive for norgestrel, boldenone, methylboldenone and methyltestosterone, respectively. These results are in agreement with concomitant in vivo experiments.

In vitro liver models are important tools to monitor the abuse of anabolic steroids in cattle.

Current veterinary residue analysis mainly focuses on the monitoring of residues of the administered parent compound. However, it is possible that larger amounts of metabolites are excreted and that they can have a prolonged excretion period. In order to unravel specific metabolic steps and to identify possible biological markers, two in vitro liver models were used, i.e. monolayer cultures of isolated hepatocytes and liver microsomes, both prepared from liver tissue of cattle. Chostebol, boldenone, norethandrolone (NE) and ethylestrenol (EES) were used as model substrates. Results show that the metabolic profiles derived from in vitro experiments are predictive for the in vivo metabolic pathways of the steroids evaluated in this study. By means of this strategy, it is possible to identify 17 alpha-ethyl-5 beta-estrane-3 alpha,17 beta-diol (EED) as a common biological marker for NE and EES. By in vivo experiments it was shown that EED is particularly important for the detection of the abuse of NE or EES because of its high excretion levels and its prolonged presence as compared with the parent compounds or any other metabolite.

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.

Interference of Helix pomatia extracts on the determination of methandriol in veterinary residue control analysis.

Aqueous solutions containing methandriol (MAD) were incubated with Succus helix pomatia (SHP) or beta-glucuronidase from Escherichia coli (EC). SHP, used for enzymatic hydrolysis of urinary steroid conjugates for residue analysis of anabolic agents, caused transformation of MAD into methyltestosterone. No conversion occurred when bacterial beta-glucuronidase from E. coli was used.

Identification of some important metabolites of boldenone in urine and feces of cattle by gas chromatography-mass spectrometry.

17 alpha-Boldenone (17 alpha-BOL) and/or 17 beta-boldenone (17 beta-BOL) appear occasionally in fecal matter of cattle. In addition to 17 alpha-BOL, a whole array of boldenone related substances can be found in the same samples. In vitro experiments with microsomal liver preparations and isolated hepatocytes combined with the excretion profiles found in urine and feces samples of in vivo experiments made it possible to identify several metabolites of 17 beta-BOL in 17 beta-BOL positive feces samples. In one animal treated with 17 beta-BOL, no 17 beta-BOL or its metabolites were present before treatment and most of these compounds disappeared gradually in time after the treatment was stopped. It is not clear what the origin is of 17 alpha-BOL and boldenone metabolites in samples screened routinely for the abuse of anabolic steroids and considered to be 'negative' because of the absence of 17 beta-BOL since other workers showed some evidence that 17 alpha-BOL can be of endogenous origin. However, in our hands, most of these 17 alpha-BOL positive samples, obtained during routinely performed screenings of cattle, contained large amounts of delta 4-androstene-3,17-dione (AED), which normally is absent from routinely screened negative samples. Furthermore, AED was absent in all samples obtained from the animals treated with 17 beta-BOL. We have no direct evidence that 17 alpha-BOL or 17 beta-BOL is of endogenous origin.

Beta-agonists in animal feed. IV: Intercomparison study of a candidate reference confirmatory method.

The objective of this intercomparison study was to evaluate the qualitative aspects and the interlaboratory performance of the method selected to be recommended as the official Community reference confirmatory method for the analysis of beta-agonists in animal feed. This method contains three possible options, i.e. a narrow range method for clenbuterol-type compounds based either on HPLC or on GCMS as the end-determination step and a broad range GCMS method for clenbuterol-type and salbutamol-type-beta-agonists. Three types of animal feed materials were provided: a series of blank materials and two series of materials contaminated with clenbuterol and salbutamol at a low and a high level, respectively. The results showed that the majority of the laboratories were able to identify blank, low and high level materials both for clenbuterol and salbutamol. For clenbuterol the narrow range GCMS method has been shown to be the most satisfactory. Although the participants had comments on the purity of the extracts obtained by means of the broad range method it was found appropriate as a multi-residue method which is able to measure simultaneously clenbuterol-type and salbutamol-type beta-agonists. A statistical evaluation of the quantitative measurement was also performed.

beta-agonists in animal feed. III: Optimization of the clean-up and the end-determination step.

This study represents the second part of an interlaboratory study intended to develop an official modular Community confirmatory method for the detection of beta-agonists in animal feed. Homogeneous pools of primary extracts were prepared by means of an extraction module based on the conclusions of a previous part of this work. The primary extracts were further processed by four laboratories each using a different clean-up scheme. The final extracts thus obtained were cross-distributed between the same laboratories and measured either by GCMS or HPLC. Two laboratories (B and D) applied separate clean-up schemes for clenbuterol and salbutamol. All clean-up schemes for clenbuterol were found to be compatible with all end-determination steps. In contrast, for salbutamol clean-up method D was found not to be compatible with the end-determination steps applied by laboratories B and C. The results of this study have clearly demonstrated that the clean-up methods for both clenbuterol and salbutamol applied by laboratory B yielded superior recoveries with an acceptable standard deviation. Therefore, in conclusion to this study, the participating laboratories recommend the clean-up schemes applied by laboratory B to serve as part of the official Community confirmatory method.

Metabolites of 4-chlorotestosterone acetate in cattle urine as diagnostic markers for its illegal use.

Seven metabolites of 4-chlorotestosterone acetate were identified in urine of cattle that received a single injection of the drug. The steroids were isolated by means of a series of clean-up steps carried out before and after enzymatic hydrolysis. The obtained extract was fractionated by high-performance liquid chromatography and each fraction was examined both by high-performance thin-layer chromatography and by capillary gas chromatography-mass spectrometry of the m-ethoxime-trimethylsilyl derivatives. The metabolites were tentatively identified by studying the mass spectra of selected peaks not found in blank samples. The structures of two metabolites, viz. 4-chloroandrost-4-ene-3,17-dione and 4-chloroandrost-4-ene-3 alpha,17 beta-diol were confirmed by chemical synthesis. The synthesized metabolites and 4-chloro-17 alpha-testosterone, a third metabolite which was identified tentatively, were located on the thin-layer chromatograms obtained. This study led to the conclusion that the illegal use of 4-chlorotestosterone acetate can be detected by identifying one or more of its metabolites in urine.

Determination of beta 2-receptor agonists in bovine urine and liver by gas chromatography-tandem mass spectrometry.

A highly specific and sensitive method for the simultaneous detection of seven beta 2-receptor agonists in bovine liver homogenates and urine was developed. A 10-g amount of liver was homogenized and treated with Subtilisin A. The resulting enzymatic digest was extracted with tert.-butanol-ethyl acetate (3:7) and the crude extract was purified on a 6-ml Bakerbond alumina neutral disposable extraction column. Subsequently, the hydrous eluate from the alumina column was buffered at pH 6 and loaded on top of a preconditioned 3-ml Bond-Elut Certify column. Urine was buffered and loaded onto a 3-ml Certify column without pretreatment. The analytes were eluted with dichloromethane-isopropanol (8:2) containing 2% ammonia. The extract obtained was trimethylsilylated and analysed by gas chromatography-tandem mass spectrometry using multiple selected reaction monitoring. The limits of detection for the beta 2-receptor agonists evaluated were between 0.5 and 5 ppb.

The identification of polyoxyethylene glycols and related compounds by capillary gas chromatography.

A sample work-up method for gas chromatographic profiling of polyoxyethylene glycol (PEG)-related compounds in pharmaceutical matrices is described. After a short sample clean-up, carbon-oxygen linkages are partially cleaved with 0.07 M boron tribromide in dichloromethane at room temperature. The reaction is stopped after 1 min by addition of 0.01 M HCl. The products are trimethylsilylated and injected onto a WCOT 50 m x 0.25 mm CP-SIL 5 CB fused silica column. Eleven model compounds, representing four common types of PEG-derivatives, have been evaluated by this method. The results show that characteristic profiles can be obtained from PEG-derivatives carrying different functional groups. Minimum detectable amounts are in the range of 200 micrograms.

Fatty acid patterns in parenterally fed premature and term infants: changes induced by intralipid and sunflower seed oil.

In order to prevent the development of essential fatty acid deficiency, 19 premature babies and term infants needing parenteral nutrition were studied during 14 days. Intralipid 20% was administered to ten and sunflower seed oil was rubbed six times daily on the skin of nine infants. Fatty acid pattern of plasma lipids were determined at birth and days 7 and 14. Levels of C16:0, C18:1, C18:2, and C20:4 did not change in the Intralipid group. In the sunflower seed oil group a decrease in C18:2 developed, which could be corrected by administering Intralipid (Intralipid was administered to four patients who had previously received sunflower seed oil during 14 days). Although Intralipid administration is often controversial or contraindicated in premature infants (hypoxia, septicemia, acidemia), it appears necessary to prevent plasmatic deficiency in C18:2. The latter cannot be prevented by topical application of sunflower seed oil, not even in very low birthweight infants.

Identification of polysaccharides in pharmaceuticals by capillary gas chromatography.

A sensitive method for the identification of polysaccharides in pharmaceuticals is described. Polysaccharides are isolated by gel filtration and subsequently hydrolysed. The monomeric carbohydrates obtained are transformed into oxime-trimethylsilyl derivatives and analysed by capillary gas chromatography. Profiles of 13 different natural or semi-synthetic polysaccharides are discussed. The profiles of the hydrolysis products can be used to identify the polysaccharides mentioned above. Possible interferences by other polymers are given. The method can be used to identify most polysaccharides used as pharmaceutical adjuvants.

Biotransformation of hexapropymate in man. Part 2: Isolation and identification of metabolites.

This paper deals with the isolation and identification of hexapropymate metabolites in man. This hypnotic drug is hydroxylated on different positions of the cyclohexane nucleus as was shown by IR, 1H-NMR and MS data of the isolated metabolites and their hydrolysis products. For the determination of the position and conformation of the metabolically introduced hydroxyl group, it was necessary to synthesize different reference compounds [12] and compare their physico-chemical data with those of the isolated metabolites. In this way the following phase-I-metabolites could be identified: 4a-hydroxy-1-(2'propynyl)cyclohexanol-1-carbamate (4a-hydroxyhexapropymate), 4e-hydroxy-1-(2'propynyl)cyclohexanol-1-carbamate (4e-hydroxyhexapropymate) and 3e-hydroxy-1-(2'propynyl)cyclohexanol-1-carbamate. The first two metabolites are also partially excreted in the form of glucuronides.