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

Ginkel, L. A. van; Stephany, R. W.; Rossum, H.J. van; Blitterswijk, H. van; Zoontjes, P.W.; Hooijschuur, R.C.M.; Zuydendorp, J.

doi:10.1016/s0378-4347(00)82886-8

Cited by 62 publications

(33 citation statements)

References 5 publications

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“…The synthetic anabolic steroid 19-nortestosterone (19NT) and its esters have been reported as widely used for growth promotion purposes in cattle. 2 The target residues generally investigated for surveillance of abuse are 17b-19NT and (primarily) the 17a-epimer in urine and bile; these analytes would be determined following incubation with hydrolytic enzymes to liberate free steroid from conjugates. [2][3][4] The evidence that the 17a-epimer is the primary metabolite in bovine is scant, since the only in vivo 19NT radiotracer study reported for cattle 5 did not involve the investigation of biotransformation products.…”

Section: Introductionmentioning

confidence: 99%

Residues and metabolism of 19-nortestosterone laurate in steers†

Sauer¹,

Howells²,

Bellworthy³

et al. 1998

Analyst

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The illegal use of nandrolone) and its esters in livestock, for growth promotion purposes, has been widely reported in the European Union. The target residues for surveillance of abuse in bovine urine and bile samples are 17a-and 17b-19NT, although this choice of target residues is not based on in vivo radiotracer biotransformation data. In this study, four steers were administered [ 3 H 2 ]-and [ 2 H 3 ] 17b-19NT laurate (2 mg kg 21 body mass) by intramuscular injection and blood, urine, faeces and bile samples were taken for 30 d until slaughter, after which tissues were sampled for total residue analysis. Total plasma radiolabelled residues reached a maximum of 56.3 ± 15.9 pmol ml 21 at 36 h and were still appreciable (13.3 ± 1.6 pmol ml 21 ) 30 d after treatment. Throughout the study period, total residue concentrations in bile (about 2-16 nmol ml 21 ), urine and faeces (0.5-3 nmol ml 21 or g 21 ) were higher than in other tissues sampled at slaughter. At slaughter there was evidence of residue accumulation in pigmented eye tissue (33.1 ± 6.1 pmol g 21 ) and in white (13.4 ± 3.4 pmol g 21 ) and black hair (28.9 ± 8.9 pmol g 21 ). Evaluation of radio-HPLC profiles of urine and bile extracts generally indicated that 19NT and 19NT laurate residues were present in relatively small amounts among a complex mixture of metabolites. GC-MS analysis of glucuronidase-hydrolysed bile extracts indicated that the major metabolites were 5b-estrane-3a,17a-diol, 5a-estrane-3b,17a-diol, 5a-estran-3a-ol-17-one (norandrosterone) and estra-1,3,5(10)-triene-3,17a-diol (17a-estradiol).

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Section: Introductionmentioning

confidence: 99%

Residues and metabolism of 19-nortestosterone laurate in steers†

Sauer¹,

Howells²,

Bellworthy³

et al. 1998

Analyst

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“…GC-MS is widely used for the quantification of a-NT in bovine bile (16,27) and the sensitivity of the assay is improved with IAC pretreatment (27). IAC purification also reduces the probability of spurious false positive results (8) immunoreactive (EIA) concentrations for a-NT in male cattle and relate these to actual (GC-MS) concentrations.…”

Section: Assay Performancementioning

confidence: 99%

“…In the EU, unofficial screening and confirmatory action limits of 2 ng/ml have been set for bovine urine (23). In Northern Ireland, bile is used for screening because concentrations of a-NT are higher than in urine (27); the 2 ng/ml limit is applied. Any sample containing more than 2 ng/ml of a-NT is analysed by GC-MS.…”

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confidence: 99%

Nortestosterone is not a naturally occurring compound in male cattle.

McEvoy

McCaughey

Cooper

et al. 1999

Veterinary Quarterly

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SUMMARY Nortestosterone (13-NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17a-epimer (a-NT). However, this also occurs naturally in pregnant cattle. It is not known whether a-NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether a-NT is naturally produced in this subset of the population. Bile samples from a total of 1,281 cattle (73 bulls and 1,208 steers) from 366 herds were collected at slaughter and initially screened by using a semi-automated EIA with multi-analyte immunoaffinity chromatography (IAC) clean-up. Bile samples from a further 38 male cattle (10 bulls and 28 steers) were analysed by high-resolution gas chromatography-mass spectrometry (GC-MS) with IAC pretreatment. Only samples containing more than 2 ng/ml a-NT were subjected to GC-MS. With 2 ng/ml a-NT as a threshold for confirmatory testing, the false positive rate of the screening EIA was 1.8%. Bulls (n = 16) and steers (n = 179) from government farms (n = 2) and which were not treated with exogenous 0-NT, did not have measurable concentrations of a-NT in their bile. Bulls (n = 35) and steers (n = 606) taken from herds (n = 204) which had no previous history of illegal growth promoter abuse also did not have a-NT in their bile. Of 32 bulls and 451 steers of unknown treatment history sampled from herds (n = 160), 56 steers from 19 herds contained GC-MS confirmed concentrations of a-NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had 0-NT-containing injection sites and five had residues of the 0-agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positivetanimals strongly suggested that exogenous 13-NT had been administered at the presenting farm. It is concluded that a-NT is not endogenous to this subset of the cattle population and that detection of this hormone in bile from bulls and steers constitutes evidence of abuse.

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“…Conventional chromatographic methods, such as gas chromatography (GC) or liquid chromatography (HPLC) coupled to mass spectrometry (MS) and tandem mass spectrometry (MS-MS), are then used as confirmatory methods because of the high specificity of the information from the molecular structure of the analyte (9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

A rapid and sensitive 384‐well microtitre format chemiluminescent enzyme immunoassay for 19‐nortestosterone

et al. 2003

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We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19-nortestosterone (19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrene microtitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier-tube-based microtitre plate reader or a sensitive back-illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra- and inter-assay CV < 10%) and accuracy (mean recovery 94-112%), with a detection limit of 0.03 ppb (1.1 x 10(-9) mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP-labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384-well microtitre plate cuts the sample/reagent volume (20 microL), a five-fold reduction with respect to the conventional 96-well microtitre plate. The developed method is suitable for high-throughput screening of 19-NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays.

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

Cited by 62 publications

References 5 publications

Residues and metabolism of 19-nortestosterone laurate in steers†

Residues and metabolism of 19-nortestosterone laurate in steers†

Nortestosterone is not a naturally occurring compound in male cattle.

A rapid and sensitive 384‐well microtitre format chemiluminescent enzyme immunoassay for 19‐nortestosterone

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