Multi-analyte approach for the determination of ng L(-1) levels of steroid hormones in unidentified aqueous samples.

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, beta-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces. The aim of this study was to develop an analytical method for the determination of ng L(-1) levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the 'black' market. For this, extraction was performed with Bakerbond C18 speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC-EI-MS2) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC-NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC-ESI-MS2). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ng L(-1) level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

Phytosterols and anabolic agents versus designer drugs.

Cholesterol is a well-known component in fats of animal origin and it also is the precursor of natural hormones. Phytosterols appear in plants and only differ slightly in structure from cholesterol. An important difference however is the low absorption in the gut of phytosterols and their saturated derivatives, the phytostanols. As a result, there is time for all kind of reactions in faecal material inside and outside of the gut. Determination of the abuse of natural hormones may be based on gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Abuse of natural hormones changes the 13C/12C ratio of some metabolites during a relatively long time. The formation of (natural) hormones in the gut may interfere with this method. Designer drugs are mainly known from sports doping. In animal fattening, designer drugs may be used as well. Small changes in the structure of (natural) hormones may lead to a new group of substances asking for new strategies for their detection and the constatation of their abuse.

Biological and chemical approaches for the detection and identification of illegal estrogens in water-based solutions.

The continuous introduction of new products used as growth promoters in animal husbandry, for sports doping and as products for body-building requires residue laboratories to initiate research on developing a strategy for the identification of 'unknown' components. In this study, a strategy is presented for elucidating the identity, the structure and the possible effects of illegal estrogenic compounds in an unidentified water-based solution. To obtain complete information on the composition and activity of the unidentified product, a multidisciplinary approach was needed. A case-study is described with a 'solution X' found during a raid. First, in vivo techniques (animal trials with mice, anatomical and histological research) were combined with in vitro techniques (the yeast estrogenic screen (YES)). In a later stage of the investigation, HPLC-fractionation, liquid chromatography-multiple mass spectrometry (LC-MSn) and gas chromatography-multiple mass spectrometry (GC-MSn) were used. Finally, the identity of 'solution X' was confirmed in a very low concentration range (10 ng/L estrone and 400 ng/l ethinyloestradiol).

Androstadienetrione, a boldenone-like component, detected in cattle faeces with GC-MS(n) and LC-MS(n).

Boldenone (1,4-androstadiene-17-ol-3-one, Bol) has been the subject of a heated debate because of ongoing confusion about its endogenous or exogenous origin when detected in one of its forms in faecal or urine samples from cattle. An expert report was recently written on the presence and metabolism of Bol in various animal species. Androstadienedione (ADD) is a direct precursor of 17beta-boldenone (betaBol). It is a 3,17-dione; ssBol is a 17-ol-3-one. Not much is published on 1,4-androstadiene-3,17-diol, which is a 3,17-diol (ADL). If animals were exposed for a longer period to one of these analytes, a metabolic pathway would be initiated to eliminate these compounds. Similar to recent testosterone metabolism studies in the aquatic invertebrate Neomysis integer, ADD, ssBol and ADL could also be eliminated as hydroxymetabolites after exposure. The presence of 11-keto-steroids or 11-hydroxy-metabolites in faecal samples can interfere with a confirmation method by gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS), after oxidation of corticosteroids with a double bond in the A-ring (e.g. prednisolone or its metabolite prednisone). The presence of androstadienetrione (ADT) in faecal samples of cattle has never been reported. The origin of its presence can be explained through different pathways, which are presented in this paper.

Determination of betamethasone and triamcinolone acetonide by GC-NCI-MS in excreta of treated animals and development of a fast oxidation procedure for derivatisation of corticosteroids.

The use of corticosteroids in combination with other hormonal substances has long been known to result in increased mass gain with bovines. Practice has demonstrated, however, that even the single use of a glucocorticoid may result in growth promoting effects. In addition to the popular dexamethasone, more recently other corticosteroids have also been misused for fattening purposes. The first part of this study deals with the detection of two of them, namely betamethasone and triamcinolone acetonide. Betamethasone was administered orally to a cow at a dose of 50 mg d-1 for 5 d, then later the same cow was injected intramuscularly with a dose of 50 mg of betamethasone dipropionate. Excretion in urine and faeces was followed with both HPLC-enzyme immunoassay and a previously described method based on negative chemical ionization mass spectrometry (NCI-MS) after oxidation. For the triamcinolone acetonide study a cow was treated with 50 mg d-1 of the drug during a 7 d period. Excretion in faeces was followed with GC-NCI-MS. As triamcinolone acetonide is resistant to the previously described oxidation procedure, however, a hydrolysis step had to be introduced prior to oxidation. In addition to this specific modification necessary for triamcinolone acetonide, in a subsequent part of this study the original oxidation procedure with pyridinium chlorochromate was re-investigated especially to shorten the procedure. With the introduction of potassium dichromate the reaction time could be decreased from 3 h to 10 min.

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.

Comparison of the possibilities of gas chromatography-mass spectrometry and tandem mass spectrometry systems for the analysis of anabolics in biological material.

Chromatographic techniques such as GC-MS play a most important role in modern multi-residue analysis of anabolic steroids. The major difference between GC-MS apparatus from different manufacturers is the way of detection and recording. Most apparatus use selected-ion monitoring (SIM) for the determination of low concentrations. Systems based on ion trap technology record in full-scan to even picogram concentrations using a computer algorithm to compare the most important peaks of the mass spectrum of the unknown to those of the standard. In this investigation the possibilities of ion trap GC-MS and the recently released GCQ MS and MS2 for the analysis of anabolics in biological material are compared.

Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces.

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the beta-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.

Determination of dexamethasone in urine and faeces of treated cattle with negative chemical ionization-mass spectrometry.

For several years, the misuse of dexamethasone and its esters in livestock production has been clearly demonstrated. The first part of the present study deals with the elaboration of a sensitive and specific method for the determination of residues of dexamethasone in excreta at the ppb level. Sample preparation for urine and faeces, including high-performance liquid chromatography (HPLC) fractionation, was carried out. The detection was based on established methodology employing negative chemical ionization-mass spectrometry (NCI-MS) after oxidation of the dexamethasone. In comparison with previous literature, the yield of oxidized dexamethasone was substantially improved and the oxidation procedure was made more simple and robust. In the second part of the study, the relationship between the dose of dexamethasone administered and the levels of the drug in excreta was investigated using this method, as was the ratio between drug levels in urine and faeces. Treatment was carried out for 7 d with an oral dose of 50 mg d-1, the maximum levels found in urine and faeces were 980 and 744 ppb, respectively. While the elimination via faeces responded much slower at the start and the end of treatment, the final part of both excretion profiles were very similar and a level of 1 ppb was reached in both matrices 9 d after the end of treatment. Gas chromatography-mass spectrometry (GC-MS) results obtained for the urine samples were compared with those obtained with direct enzyme immunoassay.

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'.