Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids.

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, ß-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L-1 for most ß-agonists to 10 µg L-1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

Applicability of an innovative steroid-profiling method to determine synthetic growth promoter abuse in cattle.

A robust LC-MS/MS method was developed to quantify a large number of phase I and phase II steroids in urine. The decision limit is for most compounds lower than 1ngml-1 with a measurement uncertainty smaller than 30%. The method is fully validated and was applied to assess the influence of administered synthetic steroids and beta-agonists on the steroidogenesis. From three animal experiments, clenbuterol, diethylstilbestrol and stanozolol, the steroid profiles in urine of bovine animals were compared before and after treatment. It was demonstrated that the steroid profiles were altered due to these treatments. A predictive multivariate model was built to identify deviations from normal population steroid profiles. The abuse of synthetic steroids can be detected in urine samples from bovine animals using this model. The samples from the animal experiments were randomly analysed using this method and predictive model. It was shown that these samples were predicted correctly in the exogenous steroids group.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

Development of a method which discriminates between endogenous and exogenous beta-boldenone.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

Determination of resorcylic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with fusarium toxins.

An EU project, FAIR5-CT-1997-3443, has been undertaken to distinguish illegal use of zeranol from consumption of food contaminated with Fusarium spp. toxin. One of the tasks was development of screening and confirmatory methods of analysis. This paper describes a new method based on two-step clean-up and GC-MS analysis. The first clean-up step is matrix-dependant; the second is applicable to both urine and meat. The MS is operated in negative chemical ionisation mode. The method is quantitative for zeranol and taleranol, alpha- and beta-zearalenol, and zearalenone and qualitative for zearalanone. Validation was performed according to the latest EU performance criteria (Commission Decision 2002/657). For analysis of urine CC(alpha) and CC(beta) for the method (microg L(-1)) were 0.06-0.11 for zeranol, 0.07-0.12 for taleranol, 0.07-0.11 for alpha-zearalenol, 0.21-0.36 for beta-zearalenol, 0.35-0.60 for zearalenone, and 0.19-0.33 zearalanone. Within-laboratory reproducibility was 16.2, 11.2, 31.9, 30.1, 26.6, and 54.2% for zeranol, taleranol, alpha-zearalenol, beta-zearalenol, zearalenone, and zearalanone, respectively. It was found that all the compounds are stable in urine at -20 degrees C for at least a year. Part of the validation program was organisation of a small proficiency study (ringtest) and a correlation study with an LC-MS-MS method developed by the Veterinary Science Division (VSD; Belfast, UK-NI). This study showed there was good correlation between results from both laboratories. The method can be used for quantitative analysis discriminating illegal use of zeranol from consumption of zearalenone-contaminated food.

Presence and metabolism of the anabolic steroid boldenone in various animal species: a review.

The review summarizes current knowledge on the possible illegal use of the anabolic steroid boldenone. The presence of' boldenone and metabolites in different animal species and the possibility of the occurrence of endogenous boldenone and metabolites is assessed, as are the methods of analysis used for detection. Different laboratories in the European Union have examined the occurrence of boldenone and its metabolites. The results were discussed at different meetings of a European Commission DG-SANCO Working Party) and summarized in an expert report. The situation of the different laboratories at this time is also covered herein. The overall conclusion of the Working Party was that there was a necessity for further research to distinguish between naturally occurring and illegally used boldenone forms. The confirmation of the presence of boldenone metabolites (free and conjugated forms) in certain matrices of animals is proposed as a marker for the illegal treatment with boldenone.

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability.

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 2: certification.

The certification by inter-laboratory testing of two candidate reference materials (RMs) for the mass concentration of the anabolic agent clenbuterol in bovine eye material is described: RM 674 with ca 10 microg clenbuterol per kg of eye matrix and RM 673 clenbuterol-free eye matrix as the negative control (<0.50 microg kg(-1)). Both candidate RMs were certified by eleven EU laboratories, and sixty-six accepted replicate measurements were included in the "Certification Study". The precision of the measurement process was assessed by calculation of the standard variation determined within each laboratory during the certification step. The study was performed according to the "Guidelines for the production and certification of BCR reference materials" and to "ISO guide 31, 33, and 35". The certified clenbuterol mass concentration for clenbuterol-free eye material CRM 673 (calculated on the basis of clenbuterol as the free base) was <0.50 microg kg(-1). The corresponding concentration for clenbuterol-containing eye material CRM 674 was 9.42 +/- 0.88 microg kg(-1). These certified values are very close to the desired target concentration of <0.5 microg kg(-1) and ca 10 microg kg(-1). This study has demonstrated that successful certification of clenbuterol-containing and clenbuterol-free bovine eye materials is possible.

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices.

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.

Supercritical fluid extraction of methyltestosterone, nortestosterone and testosterone at low ppb levels from fortified bovine urine.

A multi-residue supercritical fluid extraction (SFE) method is proposed for the isolation of nortestosterone, testosterone and methyltestosterone from bovine urine. Prior to SFE, bovine urine was hydrolyzed and then fortified with the three steroids at 100 ng/ml and 50 ng/ml each for HPLC analysis and 25 ng/ml and 12.5 ng/ml each for GC-MS analysis. The samples then were mixed with an adsorbent material, placed in an SFE extraction vessel prepacked with a 3-ml SPE column containing neutral alumina and the testosterones were extracted from the urine matrix using unmodified supercritical CO2 at 27.2 MPa and 40 degrees C. The steroids were retained in-line on the neutral alumina sorbent in the SPE column while co-extracted artifactial material was trapped off-line after CO2 decompression. After SFE, the SPE column was removed from the extraction vessel, and the trapped steroids were eluted from the neutral alumina sorbent with 3 ml of a methanol-water mixture. Eluates were used directly without post-SFE clean-up either for HPLC analysis (detection limit 50 ng/ml) or for GC-MS analysis (detection limit 5 ng/ml after steroid derivatization). The multi-residue SFE recoveries (n=6) for nortestosterone, testosterone and methyltestosterone from hydrolyzed bovine urine by GC-MS analysis were 90.8+/-6%, 93.9+/-3% and 92.5+/-5%, respectively for each steroid at the 12.5 ng fortification level.

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.

Production of CTC-containing porcine reference materials.

The European Commission (EC) established the Standards, Measurements and Testing programme for the preparation of Reference Materials (RMs) as an aid to harmonise testing for veterinary drug residues throughout the European Union (EU). The production of chlortetracycline (CTC)-free and CTC-incurred pig tissues as candidate RMs is described. High performance liquid chromatography (HPLC) with fluorescence detection of CTC and 4-epi-CTC was used for all tissue analyses. A pilot study revealed that incurred CTC residues were stable in pig kidney, liver and muscle lyophilised powders during storage for 10 weeks at -70, -20 and +37 degrees C, obviating the need for addition of a stabiliser (thimerosal). In the main study, 500 vials each of CTC-free and CTC-incurred kidney, liver and muscle were produced. Target concentrations in the CTC-incurred lyophilised tissue powders were 750-1500, 500-1000 and 300-600 micrograms kg-1 for kidney, liver and muscle, respectively. Following lyophilisation, the mean +/- s concentrations of CTC in the incurred positive RMs were 1,315 +/- 56.9, 765 +/- 35.3 and 378 +/- 16.8 micrograms kg-1 for kidney, liver and muscle respectively. Residual moisture in the RMs ranged from 1.6 +/- 0.53% for muscle to 3.0 +/- 0.50% for liver. Between-vial homogeneity for incurred powders was determined for 20 vials of each material, which had been removed at regular intervals during the filling process. Relative standard deviations (RSDs) for kidney, liver and muscle were 4.3, 4.6 and 4.4% respectively, being within the interassay RSD of the method and indicating that mixing was effective. Stability of powders stored at -18, 4, 20 and 37 degrees C was assessed over a period of 79 weeks. No measurable degradation occurred over this time period at any of the storage temperatures. It is concluded that these candidate RMs are homogenous, stable and are suitable for certification.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.

The use of supercritical fluid extraction for the determination of steroids in animal tissues.

A multi-analyte, multi-matrix method was developed for the routine determination of steroids in animal tissues (skin, meat and fat). After addition of internal standards and sample pre-treatment, the analytes of interest were extracted from the matrix with unmodified supercritical CO2 and trapped directly on an alumina sorbent placed in the extraction vessel (in-line trapping under supercritical conditions). After extraction, alkaline hydrolysis was performed and the analytes were derivatised. The samples were then analysed by gas chromatography-mass spectrometry. The limit of detection for the different matrix-analyte combinations was 2 micrograms kg-1 (for melengestrol acetate 5 micrograms kg-1), the repeatability ranged from 4 to 42% (n = 9) and the reproducibility ranged from 2 to 39% (n = 3).

Urinary cotinine and lung cancer risk in a female cohort.

In a cohort of women aged 40-64 at entry, 12 h urine samples were obtained at the beginning of a follow-up period of up to 15 years in which incident cases of lung cancer were registered as well as deaths from lung cancer. In this cohort a nested case-control study (n = 397) was carried out by measuring urinary cotinine. The method for quantitation of cotinine was sensitive enough to study lung cancer risk not only in active smokers but also in passive smokers. The results seem to indicate that cotinine estimations in single 12 h samples of urine are enough to predict lung cancer risk. Relative risk rose with increasing levels of nicotine intake already in the range associated with passive smoking. The smoking-related risk of adenocarcinoma was much less than that of other lung carcinomas.

Heterogeneity of human luteinizing hormone in pituitary tumor homogenates and cell incubates.

Incubation media of pituitary cells and homogenates of pituitary tumors were studied by isoelectric focusing followed by radioimmunoassay. Almost all LH-immunoreactive components detected had been observed in earlier studies on highly purified preparations. In one pituitary tumor homogenate, intact LH (LHi) was detected. The majority of LHi-components present in this tumor were relatively acidic (LH Type I) components. The corresponding beta-subunit population was in agreement with the one earlier observed for Type I LH. In all other tumor homogenates only alpha- and beta-subunits could be detected. In incubation media of pituitary cells from patients with no known endocrine disease nor a pituitary tumor highly acidic populations of alpha-subunits were detected. In only one of the incubation media significant amounts of LHi and free beta-subunits were observed. In an incubation medium of a TSH-producing pituitary tumor significant amounts of intact LH, free alpha- and beta-subunits were detected. The cell contents of this tumor contained components with lower pl-values than the medium. In general a good correlation is observed between the population of LH-components in material from individual identifiable pituitary glands and in highly purified preparations from large numbers of anonymous pituitary glands.

Toxicokinetics of a single intravenous dose of rac-propranolol versus optically pure propranolol in the rat.

Conscious male Wistar SPF Riv:TOX rats were dosed intravenously with 2.5, 5, or 10 mg/kg rac-propranolol.HCl, or with 5 mg/kg of either (-)-(S)- or (+)-(R)-propranolol.HCl. Disposition of (-)-(S)- and (+)-(R)-propranolol after dosing of rac-propranolol was linear in the dose range examined. Total plasma clearance was not changed in animals dosed with the individual enantiomers compared to the animals that were dosed with rac-propranolol. However, for (-)-(S)-propranolol both volume of distribution and elimination half-life decreased, whereas for (+)-(R)-propranolol increases were observed for these characteristics, in animals dosed with the individual enantiomers. Our observations suggest that the (+)-(R)-enantiomer competes with (-)-(S)-propranolol for plasma protein binding sites, resulting in lower plasma protein binding of the (-)-(S)-enantiomer when the racemate is administered. From recent toxicological experiments, it was concluded that rac-propranolol is more toxic than the individual enantiomers in the rat, when dosed iv at the same total mass. It is concluded that the observed potentiation of toxic effects of propranolol enantiomers when administered as a racemate can at least partly be explained by a pharmacokinetic interaction.

Confirmational analysis of beta-agonists by cryotrapping gas chromatography-Fourier transform infrared spectrometry.

Cryotrapping gas chromatography-Fourier transform infrared spectrometry has been used for confirmation analysis of the beta-agonists clenbuterol, salbutamol, mabuterol, bromobuterol, cimaterol, cimbuterol and mapenterol in urine and liver samples of veal calves, subsequent to selected ion detection gas chromatography-mass spectrometry. Samples have been analysed as their trimethylsilyl and methylboronate derivatives. Methylboronate derivatives yielded strongly diminished chemical background and interference levels in the infrared chromatograms of standards and samples. The limit of identification for methylboronate derivatives was at the low ppb level in incurred samples. The similarity of analyte and reference spectra, together with the retention time, was found to be a useful criterion for confirmation of unknown compounds.

Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.

High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.