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).

Rapid and high-performance analysis of thyreostatic drug residues in urine using gas chromatography-mass spectrometry.

A more sensitive method was developed using the hyphenated technique of gas chromatography-mass spectrometry (GC-MS) supplementary to the official high-performance thin-layer chromatography (HPTLC) method. Even combined with less efficient extraction and clean-up methods, GC-MS is able to lower the detection limit to less than 50 ppb. The powerful technique of GC-MS-MS is tried out to reduce the detection limit even more, in combination with simplified extraction methods. This time-saving approach combined with the increase in sensitivity is of great importance for a routine technique.

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.

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 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.

Confirmation of residues of thyreostatic drugs in thyroid glands by multiple mass spectrometry after thin-layer chromatographic screening.

A method is described for the confirmation of high-performance thin layer chromatography (HPTLC) suspect results of residues of thyreostatic drugs in thyroid tissue. The method is based on the infusion of the remainder of the extract used for HPTLC via the electrospray interface into a mass spectrometer operating in the multiple stage mass spectrometry (MSn) mode. The clean-up of the samples was performed with a selective extraction procedure, based on a specific complex formation of the drugs with mercury ions, bound in an affinity column. The thyreostatic drugs were derivatised with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

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.

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.

Endogenous occurrence of some anabolic steroids in swine matrices.

Following findings of 17beta-19-nortestosterone (150-200 microg kg(-1)) in pigs of unspecified gender imported into the European Union, a study to determine steroid and hormone levels in swine from six age/gender categories (uncastrated 'old' boars, cryptorchids, one intersex, barrows, gilts and sows) was initiated. Indeed, for some hormones there has been a discussion about their being endo- or exogenous. Tissue and urine samples from swine from each of the six categories were obtained in Belgium, France, the Netherlands and the USA. Samples were analysed in three laboratories. Quantitation was obtained for norandrostenedione, 19-nortestosterone and boldenone. The results give a well-documented overview of the status of the presence of these hormones in swine. The data illustrate that uncastrated 'old' boars produce the highest percentage of 'positive' matrices, followed by the cryptorchids. Concentrations in the matrices of the barrows and the gilts are lower. Also, sow matrices contain low amounts of nor-steroids. Furthermore, urine samples from an intersex pig contains a higher concentration of nortestosterone than sows and can therefore be suspected for illegal use of these hormones. Veterinarians taking samples in pig farms for the analysis of hormones need to be aware of the presence and concentrations of these substances in the different categories.

Detection of antibiotics in muscle tissue with microbiological inhibition tests: effects of the matrix.

The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended for muscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated with aqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solution without tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones of the beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels the differences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappeared completely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected in spiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These results indicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intact meat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initially intended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screening purposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitive for beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolones enrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.

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.

Determination of anabolic steroids with gas chromatography-ion trap mass spectrometry using hydrogen as carrier gas.

Helium is considered to be the ideal carrier gas for gas chromatography/mass spectrometry (GC/MS) in general, and for use with an ion trap in particular. Helium is an inert gas, can be used without special precautions for security and, moreover, it is needed as a damping gas in the trap. A disadvantage of helium is the high viscosity resulting in long GC run times. In this work hydrogen was tested as an alternative carrier gas for GC in performing GC/MS analyses. A hydrogen generator was used as a safe source of hydrogen gas. It is demonstrated that hydrogen can be used as a carrier gas for the gas chromatograph in combination with helium as make-up gas for the trap. The analysis time was thus shortened and the chromatographic performance was optimized. Although hydrogen has proven useful as a carrier gas in gas chromatography coupled to standard detectors such as ECD or FID, its use is not mentioned extensively in the literature concerning gas chromatography-ion trap mass spectrometry. However, it is worth considering as a possibility because of its chromatographic advantages and its advantageous price when using a hydrogen generator.

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.

Detection of corticosteroids in injection sites and cocktails by MSn.

In the European Union, the use of growth promoting substances such as thyreostats, anabolics (products with estrogenic, androgenic or gestagenic action) and beta-agonists in animal fattening is forbidden. Corticosteroids, such as dexamethasone, although considered catabolic substances, have been administered to food producing animals in order to achieve mass gains. For the analysis of injection sites and of suspect cocktails (found at the farm), a number of HPTLC and HPLC methods are used. However, in injection sites and also in cocktails found at the farm, sometimes many unknown substances are found. In this investigation, a multiple mass spectrometric (MSn) method was developed. The method is based on rapid extraction of the matrix with methanol and direct infusion of the extract into the interface of the mass spectrometer. Tables that summarise the masses of corticosteroids and their possible esters are presented.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine.

In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated.

Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues.

Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples. Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus. Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid. The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat. The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid. The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B. cereus. Two commercially available screening tests were also evaluated. The Premi(R) test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit. Tetrasensor(R), a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Determination and quantification of sulfadiazine and trimethoprim in swine tissues using liquid chromatography with ultraviolet and mass spectrometric detection.

High-performance liquid chromatographic procedures with ultraviolet detection were developed for the quantitative determination of sulfadiazine (SDA) and trimethoprim (TMP) in swine tissues (kidney, liver, muscle, fat and fat + skin). In addition, high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry was used for the confirmation of the identity of the analytes of interest. Chromatographic separation was achieved on a Spherisorb ODS-2 column (250 x 4.6 mm id, dp 5 microns). The mobile phase for SDA analysis consisted of 1% acetic acid in water-acetonitrile (85 + 15, v/v). For TMP analysis a 80 + 15 + 5 (v/v/v) mixture of 0.25% triethylammonium acetate in water, acetonitrile and methanol was used as the eluent. Sulfamerazine and ormethoprim were used as the internal standards for SDA and TMP analysis, respectively. For the isolation of the compounds of interest from biological samples, a liquid-liquid extraction with acetone and ethyl acetate, followed by a clean-up using a solid-phase extraction column (aminopropyl and benzenesulfonic acid for SDA, benzenesulfonic acid for TMP) was performed. Calibration graphs were prepared for all tissues and linearity was achieved over the concentration ranges tested (50-1000 ng g-1 for SDA, r > or = 0.9979; 25-500 ng g-1 for TMP, r > or = 0.9994). The method was validated at the maximum residue level (MRL, 100 ng g-1 for SDA and 50 ng g-1 for TMP), at half the MRL and at double the MRL for both SDA and TMP. The accuracy and precision (expressed as the within-day repeatability) were found to be within the required ranges for each specific concentration. The quantification limits were 50 ng g-1 for SDA and 25 ng g-1 for TMP. The limits of detection were below one half the MRLs. Both methods were selective for the determination of SDA and TMP. Biological samples (kidney, liver, muscle, fat and fat + skin) from pigs that received a commercial SDA-TMP preparation with the feed for five consecutive days (dose rate: 25 mg SDA and 5 mg TMP kg body weight-1 day-1) were analyzed using the described methods. The quantitative results were used to calculate a withdrawal time (12 days) to reach residue levels below the respective MRLs. This calculation was performed according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95).

Elimination profile of methylthiouracil in cows after oral administration.

In addition to methods for the determination of residues, there is an important need for knowledge of the fate and excretion of growth promoting substances in fattening animals. In court, often simple questions are asked, such as, over what period of time can the drug be detected?, is it possible to find residues after 2 months, etc. These questions can only be answered by conducting animal experiments. Data on excretion and distribution of thiouracils in cows are rather scarce. At the beginning of the 1980s, animal experiments with methylthiouracil (MTU) were carried out in the Laboratory of Chemical Analysis. These experiments showed that treatment of cows with MTU results in the rapid appearance of the drug in plasma, urine and milk, whereas MTU selectively accumulates in the thyroid gland. The results of these experiments were only published in media with limited access (thesis, abstracts) and also there has been a considerable improvement in data handling with computer programs in the last 15 years. This investigation reinterprets the 'old' analytical data from animal experiments using a pharmacokinetic software package.