A review of analytical strategies for the detection of 'endogenous' steroid abuse in food production

Drug Testing and Analysis

Volumn 4, Issue SUPPL.1, 2012, Pages 40-49

  Scarth, J P ^a   Kay, J ^b   Teale, P ^a   Akre, C ^c   Le Bizec, B ^d   De Brabander, H F ^e   Vanhaecke, L ^e   Van Ginkel, L ^f   Points, J ^a  

^a LGC   (United Kingdom)

^b Veterinary Medicines Directorate   (United Kingdom)

^c CANADIAN FOOD INSPECTION AGENCY   (Canada)

^d Waters Corporation   (France)

^e GHENT UNIVERSITY   (Belgium)

^f WAGENINGEN UNIVERSITY   (Netherlands)

Author keywords

Analysis; Endogenous; Food production; Natural; Steroids

Indexed keywords

LIPIDS; LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY;

ANALYSE; ANALYTICAL STRATEGY; ENDOGENOUS; ENDOGENOUS STEROIDS; FOOD PRODUCTION; GC/MS/MS; MODERN TECHNIQUES; NATURAL; STEROID; SYNTHETIC STEROIDS;

GAS CHROMATOGRAPHY;

19 NORETIOCHOLANOLONE; ANABOLIC AGENT; BOLDENONE; CORTICOSTEROID; ESTRADIOL; ESTROGEN; GESTAGEN; HYDROCORTISONE; NANDROLONE; NANDROLONE LAURATE; PREDNISOLONE; PROGESTERONE; STEROID; TESTOSTERONE; TRENBOLONE; TRENBOLONE ACETATE; UNCLASSIFIED DRUG;

ANALYTIC METHOD; CHEMICAL STRUCTURE; COMBINATORIAL CHEMISTRY; COMBUSTION; DRUG ABUSE; DRUG BLOOD LEVEL; DRUG DETERMINATION; DRUG FORMULATION; DRUG METABOLISM; DRUG SCREENING; DRUG URINE LEVEL; FOOD INDUSTRY; GAS CHROMATOGRAPHY COMBUSTION ISOTOPE RATIO MASS SPECTROMETRY; IMMUNOASSAY; INSTRUMENTATION; LIMIT OF DETECTION; LIQUID CHROMATOGRAPHY; LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY; LIQUID LIQUID EXTRACTION; MASS FRAGMENTOGRAPHY; METABOLITE; NONHUMAN; POLITICS; PRIORITY JOURNAL; REVIEW; SLAUGHTERHOUSE; STEROID ABUSE; STEROIDOGENESIS; TANDEM MASS SPECTROMETRY; THIN LAYER CHROMATOGRAPHY; TRANSCRIPTOMICS; ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY;

EID: 84864404375     PISSN: 19427603     EISSN: 19427611     Source Type: Journal    
DOI: 10.1002/dta.1354     Document Type: Review

References (87)

1. R.J. Heitzman. The effectiveness of anabolic agents in increasing rate of growth in farm animals; report on experiments in cattle. Environ. Qual. Saf. Suppl. 1975, 5, 89.
2. K.P. Lone. Natural sex steroids and their xenobiotic analogs in animal production: Growth, carcass quality, pharmacokinetics, metabolism, mode of action, residues, methods and epidemiology. Crit. Rev. Food Sci. Nutr. 1997, 37, 93.
3. J. Kay (ed), Analyses for Hormonal Substances in Food-Producing Animals. RSC Publishing, Cambridge, UK, 2010.
4. EU Council Directive 96/22/EC. Official Journal of the European Union. Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of beta-agonists and replacing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Off. J. Eur. Union 1996, L125, 3.
5. Scientific Committee on Veterinary Measures Relating to Public Health (SCVPH). Assessment of potential risks to human health from hormone residues in bovine meat and meat products. Available at: (Accessed 16 February 2012).
6. Scientific Committee on Veterinary Measures Relating to Public Health (SCVPH). Review of previous SCVPH opinions of 30 April 1999 and 3 May 2000 on the potential risks to human health from hormone residues in bovine meat and meat products. Available at: (Accessed 16 February 2012).
7. Veterinary Products Committee (VPC). Executive summary and critical evaluation of the scientific reasoning and methods of argument adopted in the opinion of the Scientific Committee on Veterinary Measures Relating to Public Health which assessed the potential risks to human health from hormone residues in bovine meat and meat products. Available at: (Accessed 16 February 2012).
8. Veterinary Products Committee (VPC). Risks Associated with the Use of Hormonal Substances in Food-Producing Animals. Report of the Veterinary Products Committee. Available at: (Accessed 16 February 2012).
9. EU Council Directive. Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decision 89/187/EEC and 96/664/EEC. Off. J. Eur. Union, 1996, L125, 10.
10. EU Commission. Commission Decision (2002/57/EC) of 12 August 2002. Off. J. Eur. Comm. 2002, L221, 8.
11. A.A.M. Stolker, U.A. Brinkman. Analytical strategies for analysis of veterinary drugs and growth-promoting agents in food-producing animals - a review. J. Chromatogr. A 2005, 1067, 15.
12. H. Noppe, B. Le Bizec, K. Verheyden, H.F. De Brabander. Novel analytical methods for the determination of steroid hormones in edible matrices. Anal. Chim. Acta 2008, 611, 1.
13. H.F. De Brabander, H. Noppe, K. Verheyden, J. Vanden Bussche, K. Wille, L. Okerman, et al. Residue analysis: Future trends from a historical perspective. J. Chromatogr. A 2009, 1216, 7964.
14. E. Houghton, P. Teale, E. Bichon, B. Le Bizec. Current research into new analytical procedures. in Analyses for Hormonal Substances in Food Producing Animals, (Ed. J. Kay). RSC Publishing: Cambridge, UK, 2010, pp. 171-209.
15. A.R. McKinney. Modern techniques for the determination of anabolic-androgenic steroid doping in the horse. Bioanalysis 2009, 1, 785.
16. M. Sharman, L.A. van Ginkel, J.D. MacNeil. Current analytical methods used for the detection of hormone residues, in Analyses for Hormonal Substances in Food Producing Animals, (Ed. J. Kay), RSC Publishing: Cambridge, UK, 2010, pp. 129-170.
17. L. Vanhaecke, P. Gowik, B. Le Bizec, L. Van Ginkel, E. Bichon, M. Blokland, et al. European analytical criteria: Past, present, and future. J. AOAC Int. 2011, 94, 360.
18. M.S. Moss, H.J. Rylance. A thin-layer chromatography study on the metabolism of prednisolone in the horse. J. Endocrinol. 1967, 37, 129.
19. H.F. De Brabander, J. Van Hoof. HPLC analysis of residues of anabolics in meat and kidney fat. J. Plan. Chromatogr. 1990, 3, 236.
20. B. Le Bizec, P. Marchand, D. Maume, F. Monteau, F. André. Monitoring anabolic steroids in meat-producing animals. Review of current hyphenated mass spectrometric techniques. Chromatographia 2004, 59, S3.
21. R.S. Plumb, P.D. Rainville, W.B. Potts III, K.A. Johnson, E. Gika, I.D. Wilson. Application of ultra performance liquid chromatography-mass spectrometry to profiling rat and dog bile. J. Proteome Res. 2009, 8, 2495.
22. B. Le Bizec, G. Pinel, J.-P. Antignac. Options for veterinary drug analysis using mass spectrometry. J. Chromatogr. A 2009, 1216, 8016.
23. P. Teale, E. Houghton. Metabolism of anabolic steroids and their relevance to drug detection in horseracing. Bioanalysis 2010, 2, 1085.
24. J.-P. Antignac, A. Brosseaud, I. Gaudin-Hirret, F. Andre, B. Le Bizec. Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites. Steroids 2005, 70, 205.
25. J. Scarth, P. Teale, T. Kuuranne. Drug metabolism in the horse: a review. Drug Test. Anal. 2011, 3, 19.
26. E.D. Virus, T.G. Sobolevsky, G.M. Rodchenkov. Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control. J. Mass Spectrom. 2008, 43, 949.
27. J. Scarth, H. Spencer, S. Timbers, S. Hudson, L. Hillyer. The use of in vitro technologies coupled with high-resolution/accurate-mass LC-MS for studying drug metabolism in equine drug surveillance. Drug Test. Anal. 2010, 2, 1.
28. L. Vanhaecke, K. Verheyden, J. Vanden Bussche, F. Schoutsen, H.F. De Brabander. UHPLC coupled with fourier transform orbitrap for residue analysis. LC-GC. Europe 2009, 22, 364.
29. J. Scarth, J. Roberts, P. Teale, S. Pleasance. Keeping London 2012 clean: The fight against doping in sport. Biologist 2006, 53, 305.
30. M.H. Mooney, C.T. Elliott, B. Le Bizec. Combining biomarker screening and mass-spectrometric analysis to detect hormone abuse in cattle. Trends Anal. Chem. 2009, 28, 665.
31. G. Pinel, S. Weigel, J.-P. Antignac, M.H. Mooney, C. Elliot, M.W.F. Nielen, B. Le Bizec. Targeted and untargeted profiling of biological fluids to screen for anabolic practices in cattle. Trends Anal. Chem. 2010, 29, 1269.
32. I. Riedmaier, C. Becker M.W. Pfaffl, H.H.D. Meyer. The use of omic technologies for biomarker development to trace functions of anabolic agents. J. Chromatogr. 2009, 1216, 8192.
33. J.-P. Antignac, F. Courant, G. Pinel, E. Bichon, F. Monteau, C. Elliot, et al. Mass spectrometry-based metabolomics applied to the chemical safety of food. Trends Anal. Chem. 2011, 30, 292.
34. P.M. Wynne, D.C. Batty, J.H. Vine, N.J.K. Simpson. Approaches to the solid-phase extraction of equine urine. Chromatographia 2004, 59, S51.
35. J. Scarth, C. Akre, L.A. Van Ginkel, B. Le Bizec, H.F. De Brabander, W. Korth, et al. The presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat producing animals. A review. Food Addit. Contam. A 2009, 26, 640.
36. J. Grosse, P. Anielski, P. Hemmersbach, H. Lund, R.K. Mueller, C. Rautenberg, et al. Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples. Steroids 2005, 70, 499.
37. E. Houghton, P. Teale, M.C. Dumasia. Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: The identification of urinary 19-oic acds and their decarboxylation to produce estr-4-ene-17β-ol-3-one (19-nortestosterone) and ester-4-ene-3, 17-dione (19-norandroste-4-ene-3, 17-dione) during sample processing. Anal. Chim. Acta 2007, 586, 196.
38. B. Le Bizec, F. Courant, I. Gaudin, E. Bichon, B. Destrez, R. Schilt, et al. Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle. 1. metabolite profiles of boldenone, boldenone esters and boldione in cattle urine. Steroids 2006, 71, 1078.
39. B. Destrez, E. Bichon, L. Rambaud, F. Courant, F. Monteau, G. Pinel, et al. Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldienone in cattle 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography-high resolution and tandem mass spectrometry. Steroids 2009, 74, 803.
40. G. Dervilly-Pinel, L. Rambaud, P. Sitthisack, F. Monteau, S.A. Hewitt, D.G. Kennedy, et al. 5α-Estrane-3β, 17β-diol and 5β-estrane-3α, 17β-diol: Definitive screening biomarkers to sign nandrolone abuse in cattle? J. Steroid Biochem. 2011, 126, 65.
41. R. Ventura, M. Roig, B. Pérez, S. López, M. Medina, J. Bosch, et al. Detection of the administration of 17β-nortestosterone in boars by gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 1863.
42. J. Scarth, A. Clarke, J. Hands, P. Teale, A.C. Mill, R. Macarthur, et al. Validation of an analytical biomarker approach for the detection of nandrolone abuse in the porcine. Chromatographia 2010, 72, 297.
43. R.J. Heitzman, in Veterinary Drug Residues: Residues in Food Producing Animals and their Products: Reference Materials and Methods. Blackwell Scientific Publications: Oxford, UK, 1994.
44. E. Houghton, D.L. Crone. The approaches adopted by the racing industry to address endogenous substances and substances of dietary origin, in: Proceedings of the 13th International Conference of Racing Analysts and Veterinarians, Cambridge, UK. 2000, pp. 23-26.
45. C. Aron. Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiol. Rev. 1979, 59, 229.
46. J. Scarth, A. Clarke, J. Hands, P. Teale, R. Macarthur, J. Kay. Validation of a quantitative multi-residue urinary assay for the detection of androgen, oestrogen and progestagen abuse in the bovine. Chromatographia 2010, 71, 241.
47. J. Scarth, A. Clarke, P. Teale, A.C. Mill, R. MacArthur, J. Kay. The detection of endogenous steroid abuse in cattle: results from population studies in the UK. Food Addit. Contam. A 2011, 28, 44.
48. A. Clarke, J. Scarth, J. Hands, P. Teale, A.C. Mill, R. MacArthur, et al. Detection of nandrolone and boldenone abuse in the ovine by GC-MS/MS. Chromatographia 2011, 73, 1155.
49. V. Marcos, E. Perogordo, P. Espinosa, M. Martin de Pozuelo, H. Hooghuis. Multiresidue analysis of anabolic compounds in bovine hair by gas chromatography-tandem mass spectrometry. Anal. Chim. Acta 2004, 507, 219.
50. M.W.F. Nielen, J.J.P. Lasaroms, P.P.J. Mulder, J. Van Hende, J.H.A. van Rhijn, M.J. Groot. Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry. J. Chromatogr. B 2006, 830, 126.
51. S. Boyer, P. Garcia, M.A. Popot, V. Steiner, M. Lesieur. Detection of testosterone propionate administration in horse hair samples. J. Chromatogr. B 2007, 852, 684.
52. L. Rambaud, F. Monteau, Y. Deceuninck, E. Bichon, F. André, B. Le Bizec. Development and validation of a multi-residue method for the detection of a wide range of anabolic steroids in hair using gas chromatography-tandem mass spectrometry. Anal. Chim. Acta 2007, 586, 93.
53. P. Anielski. Hair analysis of anabolic steroids in connection with doping control - results from horse samples. J. Mass Spectrom. 2008, 43, 1001.
54. A.A.M. Stolker, M.J. Groot, J.J.P. Lasaroms, A.W.J.M. Nijrolder, M.H. Blokland, I. Riedmaier, et al. Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment. Anal. Bioanal. Chem. 2009, 395, 1075.
55. E. Duffy, M.H. Mooney, C.T. Elliott, M. O'Keeffe. Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2009, 1216, 8090.
56. J.Y. Kim, M.H. Choi, S.J. Kim, B.C. Chung. Measurement of 19-nortestosterone and its esters in equine plasma by high-performance liquid chromatography with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 1835.
57. D. Hooijerink, R. Schilt, E. Van Bennekom, B. Brouwer. Determination of anabolic esters in oily formulations and plasma in husbandry using high-performance liquid chromatography and gas chromatography-mass selective detection. Analyst 1994, 119, 2617.
58. B. Gray, P. Teale, C. Pearce. Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry. Drug Test. Anal. 2011, 3, 206.
59. IFHA International Agreement on Breeding, Racing and Wagering. International Federation of Horseracing Authorities, Paris, France. Article 6. 2008. Available at: (Accessed 16 February 2012).
60. G. Balizs, A. Jainz, P. Horvatovich. Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry. J. Chromatogr. A 2005, 1067, 323.
61. P.M. Mason, S.E. Hall, I. Gilmour, E. Houghton, C. Pillinger, M.A. Seymour. The use of stable carbon isotope analysis to detect the abuse of testosterone in cattle. Analyst 1998, 123, 2405.
62. C. Buisson, M. Hebestreit, A.P. Weigert, K. Heinrich, H. Fry, U. Flenker, et al. Application of stable carbon isotope analysis to the detection of 17β-estradiol administration to cattle. J. Chromatogr. A 2005, 1093, 69.
63. V. Ferchaud, B. Le Bizec, F. Monteau, F. Andre. Determination of the exogenous character of testosterone in bovine urine by gas chromatography-combustion-isotope ratio mass spectrometry. Analyst 1998, 123, 2617.
64. V. Ferchaud, B. Le Bizec, F. Monteau, F. André. Characterization of exogenous testosterone in livestock by GC/C/IRMS: influence of feeding and age. Rapid Commun. Mass Spectrom. 2000, 14, 652.
65. M. Hebestreit, U. Flenker, C. Buisson, F. Andre, B. Le Bizec, H. Fry, et al. Application of stable carbon isotope analysis to the detection of testosterone administration to cattle. J. Agric. Food Chem. 2006, 54, 2850.
66. S. Prévost, C. Buisson, F. Monteau, F. Andre, B. Le Bizec. Is GC-C-IRMS a possible analytical approach to clear up misuse situations for forbidden natural substances in edible tissues? in Proceedings of Euroresidue 5. Noordwijkerhout, The Netherlands, Vol 2 2004, 777.
67. T. Piper, H. Geyer, V. Gougoulidis, U. Flenker, W. Schänzer. Determination of 13C/12C ratios of urinary excreted boldenone and its main metabolite 5β-androst-1-en-17β-ol-3-one. Drug Test. Anal. 2010, 2, 217.
68. ISOSTER project report GRD-2001-40085 executive summary. Determination of the origin of hormones in cattle. Coordinated by the Bundesinstitut fur Risikobewetung. 2006. (Accessed 10 April 2012).
69. C. Saudan, N. Baume, N. Robinson, L. Avois, P. Mangin, M. Saugy. Testosterone and doping control. Br. J. Sports Med. 2006, 40, i21.
70. M.E. Dumas, C. Canlet, F. André, J. Vercauteren, A. Paris. Metabonomic assessment of physiological disruptions using 1H-13C HMBC/NMR spectroscopy combined with pattern recognition procedures performed on filtered variables. Anal. Chem. 2002, 74, 2261.
71. M.E. Dumas, L. Debrauwer, L. Beyet, D. Lesage, F. André, A. Paris, et al. Analysing the physiological signature of anabolic steroids in cattle urine using pyrolsysis/metastable atom bombardment mass spectrometry and pattern recognition. Anal. Chem. 2002, 74, 5393.
72. I. Riedmaier, A. Tichopad, M. Reiter, M.W. Pfaffl, H.H.D. Meyer. Identification of potential gene expression biomarkers for the surveillance of anabolic agents in bovine blood cells. Anal. Chim. Acta 2009, 638, 106.
73. G. Cacciatore, S.W.F. Eisenberg, C. Situ, M.H. Mooney, P. Delahaut, S. Klarenbeek, et al. Effect of growth-promoting 17β-estradiol, 19-nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves. Anal. Chim. Acta 2009, 637, 351.
74. S. Anizan, D. Di Nardo, E. Bichon, F. Monteau, N. Cesbron, J.-P. Antignac, et al. Targeted phase II metabolites profiling as new screening strategy to investigate natural steroid abuse in animal breeding. Anal. Chim. Acta 2011, 700, 105.
75. M. Groot. PhD thesis. Histological Screening for illegal administration of growth promoting agents in veal calves. Rijksuniversiteit: Utrecht, The Netherlands, 1992. ISBN: 90-393-0361-4.
76. M.J. Groot, J.S. Ossenkoppele, R. Bakker, M.W. Pfaffl, H.H.D. Meyer, M.W.F. Nielen. Reference histology of veal calf genital and endocrine tissues - an update for screening on hormonal growth promoters. J. Vet. Med., Ser. A 2007, 54, 238.
77. P. Van Renterghem, P. Van Eenoo, P.-E. Sottas, M. Saugy, F. Delbeke. Subject based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports. Drug Test. Anal. 2010, 2, 582.
78. C.J.M. Arts, M.J. Van Baak, H. Van Der Berg, R. Schilt, P.L.M. Berende, J.M.P. Den Hartog. Concentrations of the endogenous steroid hormones oestradiol-17beta, testosterone and progesterone in veal calves in connection with the control for illegal administration. Archiv fur Lebensmittelhygine 1990, 41, 58.
79. H.F. De Brabander, J. Vanden Bussche, W. Verbeke, L. Vanhaecke. The economics of residue analysis. TrAC, Trends Anal. Chem. 2011, 30, 1088.
80. G.D. Kennedy, H. Desmond Shortt, S.R.H. Crooks, P.B. Young, H.J. Price, W.G. Smyth, et al. Occurrence of α- and β-nortestosterone residues in the urine of injured male cattle. Food Addit. Contam., A 2009, 26, 683.
81. F. Arioli, M. Fidani, A. Casati, M.L. Fracchiolla, G. Pompa. Investigation on possible transformations of cortisol, cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography-mass spectrometry. Steroids 2010, 75, 350.
82. K. Verheyden, H. Noppe, J. Vanden Bussche, K. Wille, K. Bekaert, L. De Boever, et al. Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE®) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS/MS). Anal. Bioanal. Chem. 2010, 397, 345.
83. R. Draisci, C. Montesissa, B. Santamaria, et al. Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression. Proteomics 2007, 7, 3184.
84. M.E. Dumas, C. Canlet, L. Debrauwer, P. Martin, A. Paris. Selection of biomarkers by a multivariate statistical processing of composite metabonomic data sets using multiple factor analysis. J. Proteome Res. 2005, 4, 1485.
85. J.C. Rijk, A. Lommen, M.L. Essers, M.J. Groot, J.M. Van Hende, T.G. Doeswijk, et al. Metabolomics approach to anabolic steroid urine profiling of bovines treated with prohormones. Anal. Chem. 2009, 81, 6879.
86. G. Dervilly-Pinel, S. Weigel, A. Lommen, S. Chereau, L. Rambaud, M. Essers, et al. Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves. Anal. Chim. Acta 2011, 700, 144.
87. P. Regal, S. Anizan, J.-P. Antignac, B. Le Bizec, A. Cepeda, C. Fente. Metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry to screen for the illegal use of estradiol and progesterone in cattle. Anal. Chim. Acta 2007, 700, 16.