Past, present and future of mass spectrometry in the analysis of residues of banned substances in meat-producing animals.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Banned substances, such as growth promoters, which are abused in animal fattening and where this article is focused on, may be divided into four major groups: thyreostats, anabolics or anabolic steroids, corticosteroids and beta-agonists or repartitioning agents. The combination of chromatographic techniques with mass spectrometry (GC-MS(n), LC-MS(n), etc.) plays a key role in the production of specific results in residue analysis. In this review, the past, present and future of mass spectrometry in this area are discussed in the light of the impact of these substances on human health and the reliable production of analytical results, ready for challenge in a court.

Metabolism of methenolone acetate in a veal calf.

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, alpha-methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and 3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. Alpha-methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.

New anabolic steroid illegally used in cattle-structure elucidation of 19-norchlorotestosterone acetate metabolites in bovine urine.

4-Chloro-estr-4-en-17-ol-3-one, trivially named 19-norclostebol acetate or 4-chloro-19-nortestosterone acetate (NClTA), has been identified on the European black market in the late 1990s for possible use in breeding animals. After oral and subcutaneous administration of NClTA to bovine, urine samples were collected over a period of three weeks, and chemical structure of main excreted urinary metabolites was determined. After oral administration, the most abundant metabolites were mainly reduced as 4-chloro-19-norandrostan-3xi-ol-17-one and 4-chloro-19-norandrostan-3xi,17xi-diol. They were identified until 1 week after administration. Following subcutaneous injection, 4-chloro-19-norandrostan-3xi-ol-17-one was again of major abundance, but so were 4-chloro-19-norandrost-4-ene-3xi,17xi-diol and 4-chloro-19-norandrost-4-en-3xi-ol-17-one. They were detected at least 3 weeks after administration. Whatever the route of administration, metabolites were found mainly glucurono-conjugated; the only exception was metabolite 4-chloro-19-norandrostan-3xi-ol-17-one which was identified both in the sulpho- and glucurono-fractions.

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).

Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry.

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.

Determination of mercaptobenzimidazol and other thyreostat residues in thyroid tissue and meat using high-performance liquid chromatography-mass spectrometry.

This paper describes a method for extraction of tapazol, thiouracil, methylthiouracil, propylthiouracil and mercaptobenzimidazol (MBI) from thyroid tissue. The solid-phase extraction procedure is optimized to obtain the maximum results for the main thyreostats including MBI. Different combinations of sample application, column conditioning and wash steps were tested. The analytes were extracted from the matrix with methanol. After solid-phase extraction they were derivatised with 7-chloro-4-nitrobenzo-2-furazan. Determination is carried out using liquid chromatography-electrospray mass spectrometry. The identification of the analytes was performed according to the final revision of the EU criteria (93/256/EC decision). The detection capability was 20 microg kg(-1) for all mentioned thyreostats.

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.

Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry.

The objective of this study was to provide LC and GC-multiple mass spectrometry (MSn) data in positive and negative ion modes to prove the distinction between dexamethasone and betamethasone in a mixture of both components. Using GC-MS, the differentiation was based on a difference in the ratio of the ion traces of the two chromatographic peaks of the alpha and beta epimer with m/z 310 and 330. A minimum of 15% dexamethasone should be present in a mixture of both to detect it as present with a probability of 95%. In the same way betamethasone can be detected from 15% on. Because of the very similar structures of the dexamethasone and betamethasone epimers, no reversed-phase (RP) separations have been reported. Normal-phase separations have been reported in other studies. However because of the compatibility of RP mobile phases in the coupling with MS, the latter was the method of choice. In LC-MSn positive ion mode the product ion 355 was plotted against the sum of 337 and 319. With this combination dexamethasone and betamethasone could be discriminated in a mixture of 20 to 80% of each combination of analytes. In negative ion mode only two product ions were formed from the fragmentation of the acetate adduct, [M-H]- and [M-H-CH2O]-. The intensity of the fragment 391 ([M-H]-) was determined in the discrimination of the two epimers.

Preliminary study on the presence of prednisolone in porcine urine and liver - How to distinguish endogenous from therapeutically administered prednisolone.

In animal breeding in Europe, synthetic corticosteroids are not allowed as growth-promoting agents. However, prednisolone residues have recently been found in porcine urine samples collected at slaughterhouses. The aim of this work was therefore to look for prednisolone in porcine urine and liver, to determine if detected residues might be of endogenous origin, and to check the possible relation with stress. An analytical method developed in-house was validated, combining immunoaffinity-based purification and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). This method was applied to urine and liver samples collected from sows experimentally treated either with prednisolone or tetracosactide hexaacetate (synthetic analogue of ACTH). Thanks to the performance of the analytical method, both cortisol and prednisolone were detected in all pig urine samples collected before or after administration of prednisolone or tetracosactide hexaacetate. High levels of prednisolone were found in porcine urine just after prednisolone administration, decreasing quickly to within the range detected in non-treated animals. In urine, the cortisol level varied depending on the time lapse between administration and sampling. On the other hand, prednisolone was detected also in liver samples of treated pigs. In this matrix, the cortisol level remained constant and prednisolone/cortisol level could be used to detect prednisolone administration at least 4 days after injection. In conclusion, the best indicator for detecting illicit prednisolone administration to pigs seems to be the prednisolone/cortisol ratio in liver samples. This preliminary work must be confirmed by a larger-scale study and metabolites should also be included.

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.

Mass spectrometric detection of and similarities between 1-androgens.

Regularly new anabolic steroids appear on the black market. In most cases these substances are marketed on websites or are confiscated during inspections. 1,(5alpha)-Androstene-17beta-ol-3-one, also known as 1-testosterone, is one of these substances presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone closely resembles the natural hormone testosterone except for a 1,2-double bound instead of a 4,5-double bound. 1-Androstene-3beta,17beta-diol is transformed into 1-testosterone after oral administration. 1-Testosterone, 1-androstene-3beta,17beta-diol and some other related 'new' anabolic steroids were studied with gas chromatography coupled to mass spectrometry (GC-MS) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS2) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the interpretation of the derivatised analytes, are discussed.

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.

High-performance liquid chromatographic determination of clenbuterol and cimaterol using post-column derivatization.

A general high-performance liquid chromatographic method for the simultaneous and rapid determination of cimaterol and clenbuterol is described. Solid samples, such as animal tissues, faeces and feeding-stuffs, are extracted with dilute acid saturated with ethyl acetate. The resulting extracts or liquid samples, such as urine, plasma, blood and bile, are purified via Chem Elut columns. Separation is achieved by ion-pair chromatography on a Nova-Pak C18 column, and highly specific detection is obtained with an adapted version of the post-column derivatization described previously for the determination of clenbuterol in urine and animal tissues. Detection limits for liquids and solids are 0.1 ng/ml and 0.2 ng/g, respectively. The results are in complete agreement with analysis by high-performance thin-layer chromatography and gas chromatography-mass spectrometry, applied for confirmation after the same sample pretreatment. With this simple method, complete analysis of a liquid sample needs about 30 min and, even without an automatic sampler, 40 samples can be completely analysed in one day. This method has been used on a routine scale for nearly two years.

Beta-agonists in animal feed. I: First intercomparison of methods of analysis for clenbuterol in animal feed materials.

The European Commission, Measurement and Testing Programme (BCR) has initiated a project to improve the methodology for analysis of beta-agonists in animal feeding stuffs. An intercomparison of methods for clenbuterol in animal feed is described. The study involved 13 European laboratories which analysed a blank feed and three feed samples with three different levels of clenbuterol contamination. The participants used a variety of extraction (organic or aqueous solvents), clean-up (liquid-liquid, silica and C-18 solid phase and immuno-affinity chromatography) and end-point detection (HPLC, GC-MS and TLC) steps. The purpose of this study was to identify and to quantify clenbuterol. The coefficient of variation from all the results for the low level (25 micrograms/kg) was 39%, for the intermediate level (100 micrograms/kg) 52% and for the high level (1000 micrograms/kg) 35%. The study showed that the initial extraction, the modular clean-up step and their compatibility to the HPLC and the GC-MS determination step were critical steps.

Beta-agonists in animal feed. II: Optimization of the extraction.

New projects of the European Commission, Measurement and Testing Programme (BCR) were set up in order to develop a modular sample preparation system for the determination of beta-agonists and animal feeds. Three phases are included: an extraction study, a clean-up study and finally a Second Intercomparison. This paper describes the extraction study in which four laboratories were involved. A total of 33 extraction conditions were tested regarding their yield on clenbuterol and salbutamol, their compatibility towards several clean-up and chromatographic end-methods and the influence of undesired coextractives. The conditions differed with respect to five factors: with or without organic solvent, temperature, pH, agitation and centrifugation. Their influence was examined via a ruggedness-test approach. A unique set-up allowed the combination of individual results in a complete factorial design. The addition of an organic solvent was found to be the most important factor. Interactions between factors were also studied. The best combinations of factors regarding the extraction are given. Finally limits for applicability and influence of organic solvents, pH and temperature were evaluated in a fifth laboratory towards enzyme immunoassay as detection method.

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.

LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver.

European legislation forbids the use of beta-agonists as growth-promoting substances in cattle raised for human consumption. However, the use of beta-agonists is allowed as a therapeutic treatment of tocolysis for female cattle during calving and of respiratory diseases and tocolysis for horses not raised for human consumption. A maximum residue limit (MRL) of 0.5 microgram kg-1 for clenbuterol in the liver of cattle and horses is proposed by law. Residues of beta-agonists in liver are identified with LC-MS-MS. Using ion trap technology, it was possible to identify each analyte without the need to resolve completely the chromatographic peaks. For each analyte, specific fragment ion spectra were obtained. The coeluting or incompletely resolved peaks were separated mass spectrometrically. For tulobuterol, bromobuterol and mabuterol, qualitative information was obtained. All beta-agonists could be detected up to a concentration of 0.1 microgram kg-1. For clenbuterol, a limited quantitative validation was performed. A working range was defined for which the method was applicable. Quantification was based on the integration of the response of the analytes in spiked blank liver samples. The mean recovery was 15%. The relative standard deviation (RSD) values at different concentrations were below the maximum allowed RSD. The limit of detection of clenbuterol was 0.11 microgram kg-1. The limit of quantification was 0.21 microgram kg-1. It was possible to quantify clenbuterol below one-half of the MRL. The advantage of this method is the ease of use of the mass spectrometric separation to qualify and quantify the presence of four beta-agonists in liver.

Clenbuterol residue analysis by HPLC-HPTLC in urine and animal tissues.

A method for clenbuterol residue analysis in urine and animal tissues has been developed. The detection limits are 0.25 micrograms/l and 0.5 micrograms/kg, respectively. The recovery in urine varies from 85% to 90% and in animal tissues from 70% to 74%. The beta 2-agonist was liberated from the tissues by an enzymatic digestion, purified on Chem Elut columns using alkaline conditions and extracted with 0.01 mol/l HCl. Clenbuterol was quantified by high-performance liquid chromatography (HPLC) on a RP-8 column and a post-column reaction procedure. High-performance thin-layer chromatography (HPTLC) was performed on silica gel 60 plates and clenbuterol visualized by means of the modified Ehrlich's TLC spray reagent. Since this method is sensitive, as is HPLC, it was used to obtain a confirmation and to exclude false positive results.