P.W. Zoontjes

Effective monitoring of residues of nortestosterone and its major metabolite in bovine urine and bile.

The results of a newly developed method for the detection and identification of residues of nortestosterone (NT) and one of its major metabolites, 17 alpha-nortestosterone (epiNT) are described. The method is based on sample clean-up by immunoaffinity chromatography and detection by high-performance liquid chromatography and/or gas chromatography-mass spectrometry (selected-ion monitoring). All samples of bile from calves that had been treated with NT contained significant amounts of epiNT (6-18 micrograms/l). The NT content of these samples, if detectable, was below 1 microgram/l. Urine contained, with one exception, less than 1 microgram/l epiNT. NT itself if detectable, was, present in urine or bile at levels below 0.1 microgram/l. The results corresponds well with results obtained with a radioimmunoassay procedure.

Assay for trenbolone and its metabolite 17α-trenbolone in bovine urine based on immunoaffinity chromatographic clean-up and off-line high-performance liquid chromatography-thin-layer chromatography

An high-performance liquid chromatography (HPLC)-thin-layer chromatography (TLC) method was developed to detect the illegal use of the xenobiotic growth promotor Trenbolone acetate (TBA). Very effective clean-up of bovine urine was achieved by immunoaffinity chromatography (IAC). The active form of TBA, the steroid 17 beta-Trenbolone (17 beta-TB), as well as its major metabolite 17 alpha-Trenbolone (17 alpha-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17 alpha-TB and 17 beta-TB (TB-fraction) was collected, and for confirmation 17 beta- and 17 alpha-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1-2 micrograms TB/l. Off-line TLC detection was even more sensitive, 0.5 microgram 17 beta- or 17 alpha-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17 beta- and 17 alpha-TB was clearly demonstrated. A survey of the illegal use of TBA in cattle was performed by applying the assay to urine obtained at slaughter. No residues of TBA or its metabolites were found in any of the 144 random samples from the Dutch public health surveillance programme.

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).

Determination of acetyl gestagenic steroids in kidney fat by automated supercritical fluid extraction and liquid chromatography ion-trap mass spectrometry

Acetyl gestagenic steroids are isolated from animal tissues such as bovine kidney fat by automated supercritical fluid extraction (SFE). After the addition of internal standards and sample pretreatment, the analytes are extracted from the matrix by supercritical CO2 and trapped directly in-line on alumina placed in the extraction vessel. The samples are analysed by liquid chromatography combined with ion-trap mass selective detection (LC-MSn). For quantification, deuterated internal standards are added and single ions of the analytes and internal standards are monitored. For confirmation of the identity of the analytes, two transition ions (one MS2 and one MS3) were monitored and the ratios between the ions were calculated and compared with those of standards. The detection capability for the multi-analyte LC-MSn analysis of megestrol acetate (MA), medroxyprogesterone acetate (MPA), chlormadinone acetate (CMA) and melengestrol acetate (MGA) is 0.5 microg kg(-1). The mean within-laboratory reproducibility ranges from 16-19% (%RSD) at a concentration level of 0.5 microg kg(-1) (n = 9). Running the SFE procedure overnight allows the analysis of 24 samples of fat per day.

Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

Abstract A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [ 37 Cl 2 ]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 μg kg −1 are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices.

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.

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.

Investigation of the presence of prednisolone in bovine urine

Over the past two years low levels of prednisolone have been reported in bovine urine by a number of laboratories in European Union member states. Concentrations vary, but are reported to be below approximately 3 µg l–1. Forty per cent of bovine urine samples from the Dutch national control plan had concentrations of prednisolone between 0.11 and 2.04 µg l–1. In this study the mechanism of formation of prednisolone was investigated. In vitro conversion of cortisol by bacteria from faeces and soil, bovine liver enzymes and stability at elevated temperatures were studied. In vitro bovine liver S9 incubation experiments showed a significant 20% decrease of cortisol within 6 h, and formation of prednisolone was observed from 0.2 g l–1 at t = 0 to 0.5 g l–1 at t = 6. Under the influence of faeces, the stability of cortisol in urine is reduced and cortisol breaks down within 50 h. Prednisolone is formed up to 4 µg l–1 at 70°C after 15 h. However, this decreases again to zero after 50 h. With soil bacteria, a slower decrease of cortisol was observed, but slightly higher overall formation of prednisolone, up to 7 µg l–1 at 20°C. As opposed to incurred urine, in fortified urine incubated with faeces or soil bacteria no prednisolone was detected. This difference may be explained by the presence of natural corticosteroids in the incurred sample. With UPLC-QToF-MS experiments, in urine and water samples incubated with faeces, metabolites known from the literature could be (tentatively) identified as 20β-hydroxy-prednisolone, cortisol-21-sulfate, oxydianiline, tetrahydrocortisone-3-glucuronide and cortexolone, but for all compounds except 20β-hydroxy-prednisolone no standards were available for confirmation. Based on the results of this study and literature data, for regulatory purposes a threshold of 5 µg l–1 for prednisolone in bovine urine is proposed. Findings of prednisolone in concentrations up to 5 µg l–1 in bovine urine can, most likely, originate from other sources than illegal treatment with growth promoters.

Perspectives in residue analysis; The use of immobilized antibodies in (multi) residue analysis

Abstract The use of immobilized antibodies in residue analysis is rapidly increasing. An overview of one of the interesting areas, the use of immunoaffinity chromatography, is given. A distinction is made between: (1) single antibody, single analyte procedures; (2) single antibody, multi-analyte procedures; and (3) multi-antibody, multi-analyte procedures. We conclude that immunoaffinity chromatography is superior to most other techniques of sample preparation and extract clean-up. In comparison with, e.g., high-performance liquid chromatography the advantages are clearest in multi-residue procedures. In combination with gas chromatography low resolution mass spectrometry, very effective multi-residue methods are possible.