Identification of in vitro metabolites of boldenone using Camel liver microsomes – A HR-LCMS approach

Abstract views: 205 / PDF downloads: 359


  • RAMEES ABDULLA VAZHAT Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates
  • N A MOHAMED FAROOK Khadir Mohideen College, Bharathidasan University, Adirampattinam, Tamil Nadu
  • J NALAKATH Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates
  • PRASEEN O K Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates


Anabolic steroids, Biotransformation, Boldenone, Camel liver, In vitro metabolism, HRMS


Anabolic steroids are widely abused in animal sports to improve their performance. The present study identifies the possible metabolites of boldenone in camel liver. A high-resolution accurate QE mass spectrometer was used to identify the parent boldenone and its metabolites. To investigate the phase 1 biotransformation of boldenone in camel, sodium phosphate buffer and NADPH were used. Chromatographic separation was carried out on a Thermo Hypersil C18 column using acetonitrile and formic acid as mobile phases. The current study, helped in unequivocal detection of six metabolites (Phase 1) for boldenone. The 17b-boldenone is an 3-oxo-Delta (1), Delta(4)-steroid substituted by an oxo group at position 3 and a beta-hydroxy group at position 17. It is prone to oxidation, which results in three hydroxylated metabolites with protonated parent ion of m/z 303.1954 (C19H27O3)+. Androsta-1,4-diene-3,17-dione [M+H]+ of m/z 285.1848 (C19H25O2+), hydroxyandrosta-1,4-diene-3,17-dione [M+H]+ of m/z 301.1797 (C19H25O3+) in addition to 17-hydroxy-androsta-1-en-3-one [M+H]+ of m/z 289.2162 (C19H29O2+) were also identified. The structures of the detected metabolites were identified based on their accurate mass, fragmentation pattern, and chromatographic retention time. In this research, camel liver was successfully used for in vitro experiments as an alternative to liver microsomes.


Download data is not yet available.


Al Katheeri N A, Wasfi I A, Lambert M, Giuliano Albo A and Nebbia, C. 2006. In vivo and in vitro metabolism of dexamethasone in the camel. Veterinary Journal 172(3): 532–43. DOI:

Balcells G, Pozo O J, Esquivel A, Kotronoulas A, Joglar J, Segura J and Ventura R. 2015. Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry. Journal of Chromatography A 1389: 65–75. DOI:

Brandon E F A, Raap C D, Meijerman I, Beijnen J H and Schellens J H M. 2003. An update on in vitro test methods in human hepatic drug biotransformation research: Pros and cons. Toxicology and Applied Pharmacology 189(3): 233–46. DOI:

Buiarelli F, Cartoni G P, Coccioli F, Giannetti L, Merolle M, Neri B and Terracciano A. 2005. Detection of boldenone and its major metabolites by liquid chromatography — tandem mass spectrometry in urine samples. Analytica Chimica Acta 552: 116–26. DOI:

Cannizzo F T, Zancanaro G, Spada F, Mulasso C and Biolatti B. 2007. Pathology of the testicle and sex accessory glands following the administration of boldenone and boldione as growth promoters in veal calves. Journal of Veterinary Medical Science 69(11): 1109–16. DOI:

De Brabander H F, Poelmans S, Schilt R, Stephany R W, Le Bizec B, Draisci R, Sterk S S, van Ginkel L A, Courtheyn D, Van Hoof N, Macrì A and De Wasch K. 2004. Presence and metabolism of the anabolic steroid boldenone in various animal species: A review. Food Additives and Contaminants 21(6): 515–25. DOI:

Fred Hartgens, Wouter D. Van Marken Lichtenbelt, Spike Ebbing, Niels Vollaard, Gerard Rietjens and Harm Kuipers. 2001. Androgenic-anabolic steroid—induced body changes in strength athletes. The Physician and Sportsmedicine 29(1): 49–46. DOI:

IARC I A for R on C. 1987. Evaluation of carcinogenic risks. Overall evaluations of carcinogenicity: IARC Monographs, 1–42.

Mayada R F, Taghred M S and Haytham A A. 2015. Boldenone-induced apoptotic, structural, and functional alterations in the liver of rabbits. World Rabbit Science 23(1): 39–46. DOI:

Merlanti R, Gallina G, Capolongo F, Contiero L, Biancotto G, Dacasto M and Montesissa C. 2007. An in vitro study on metabolism of 17β-boldenone and boldione using cattle liver and kidney subcellular fractions. Analytica Chimica Acta 586(1-2 SPEC. ISS.): 177–83. DOI:

Nielen M W F, Rutgers P, Van Bennekom E O, Lasaroms J J P and Van Rhijn J A. 2004. Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17- dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 801(2): 273–83. DOI:

Philip M, Karakka Kal A K, Subhahar M B, Karatt T K, Mathew B and Perwad Z. 2022. In vitro studies of hypoxia inducible factor-prolyl hydroxylase inhibitors daprodustat, desidustat, and vadadustat for equine doping control. Drug Testing and Analysis 14(2): 317–48. DOI:

Philip M, Karakka Kal A K, Subhahar M B, Perwad Z and Karatt T K. 2021. Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren. Rapid Communications in Mass Spectrometry 35(23). DOI:

Philip M, Mathew B, Karatt T K, Perwad Z, Subhahar M B and Karakka Kal A K. 2021. Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control. Drug Testing and Analysis 13(4): 794–816. DOI:

Popot M A, Stojiljkovic N, Garcia P, Bonnaire Y and Tabet J C. 2003. First mass spectrometric detection of boldenone in horse mane samples. Chromatographia 57(3–4): 255–60. DOI:

Schanxer W and Domke M. 1993. Metabolism of anabolic steroids in man: synthesis and use of reference substances for identification of anabolic steroid metabolites. Analytica Chimica Acta 275: 23–48. DOI:

Soma L R, Uboh C E, Guan F, Mcdonnell S and Pack J. 2007. Pharmacokinetics of boldenone and stanozolol and the results of quantification of anabolic and androgenic steroids in race horses and nonrace horses. Journal of Veterinary Pharmacology and Therapeutics 30(2): 101–08. DOI:

Van Puymbroeck M, Kuilman M E M, Maas R F M, Witkamp R F, Leyssens T, Vanderzande D, Gelan J and Raus J. 1998. In vitro liver models are important tools to monitor the abuse of anabolic steroids in cattle. Analyst 123(12): 2453–56. DOI:

Van Puymbroeck Mark, Kuilman M E M, Maas R F M, Witkamp R F, Leyssens L, Vanderzande D, Gelan J and Raus J. 1998. Identification of some important metabolites of boldenone in urine and feces of cattle by gas chromatography-mass spectrometry. Analyst 123(12): 2681–86. DOI:

Verheyden K, Noppe H, Zorn H, Van Immerseel F, Bussche J Vanden, Wille K, Bekaert K, Janssen C R, De Brabander H F and Vanhaecke L. 2010. Endogenous boldenone-formation in cattle: Alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle’s feed. Journal of Steroid Biochemistry and Molecular Biology 119(3–5): 161–70. DOI:









How to Cite

VAZHAT, R. A., FAROOK, N. A. M., NALAKATH, J., & K, P. O. (2023). Identification of in vitro metabolites of boldenone using Camel liver microsomes – A HR-LCMS approach. The Indian Journal of Animal Sciences, 93(1), 39–44.