Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry
Fuyu Guan
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1
2
3
Lawrence R. Soma
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1
2
3
Yi Luo
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1
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Cornelius E. Uboh
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Thermo Electron Corporation, Somerset,
New Jersey, USA
1
Pennsylvania Equine Toxicology and Research Center, West Chester University
, West Chester,
Pennsylvania, USA
2
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania
, Kennett Square,
Pennsylvania, USA
3
Published online February 17, 2006 Part of this article was presented at the 53rd ASMS Conference on Mass Spectrometry, June 5-9, 2005, San Antonio,
TX, USA
. Address reprint requests to Dr. Cornelius E. Uboh,
PA Toxicology and Research Center
, 220 East Rosedale Ave., West Chester,
PA 19382, USA
Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MSn) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication [19]. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites. (J Am Soc Mass Spectrom 2006, 17, 477- 489) 2006 American Society for Mass Spectrometry
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Athe male sex hormones (androgens), and are used
nabolic steroids are synthetic substances related to
for the treatment of metabolic disorders in man
and animals. These agents can be illegally used to enhance
performance in human and animal sports including horse
racing [13]. The misuse of anabolic steroids is well
documented; for example, the scandal of the designer
drug tetrahydrogestrinone (THG) [4]. Anabolic steroids
are prohibited for use by athletes in competition by the
International Olympic Committee (IOC) [5], classified as
controlled substances by the United States Drug
Enforcement Agency, included under the Drugs of Misuse Act by
the United Kingdom, and banned by the European Union
for use in agricultural animals [6, 7]. Methods for
qualitative and quantitative analysis of anabolic steroids based
on gas chromatography integrated with mass
spectrometry (GC-MS) or liquid chromatography coupled with mass
spectrometry (LC-MS) have been reported [8 12].
Essential to the published LC-MS methods is selected reaction
monitoring (SRM) based on tandem mass spectrometric
(MS/MS) product ions. Interpretation of product-ion
spectra of anabolic steroids and exploration of their
collision-induced dissociation (CID) pathways are of
importance to mass spectrometric characterization of new
designer steroids, and helpful in understanding specificity
of product ions of anabolic steroids for their detection,
identification, and confirmation.
While interpretation of mass spectra obtained under
electron ionization (EI) conditions is well understood and
documented [13, 14], interpretation of product-ion spectra
of [M H] ions acquired under atmospheric pressure
ionization (API) MS/MS conditions is generating new
D OH
C 20H28O2, MW 300.2
Methandrostenolone
OH O
OH O OH O
C 19H28O2, MW 288.2
Normethandrolone
Chemical structures of anabolic steroids used in this study.
interest. Fragmentation pathways are reported for
testosterone and testosterone hydroxyl analogs [15], oxosteroid
Girard derivatives [16, 17], and steroid oximes [18] under
ESI conditions. Recently, Thevis et al. reported
electrospray ionization tandem mass spectrometric
characterization of chemically modified steroids including 21
gestrinone and testosterone analogs [19]. In this paper,
formation of major product ions of selected anabolic
steroids under ESI ( ) MS/MS conditions are interpreted
and fragmentation pathways are proposed for those
steroids of which fragmentation pathways have not been
previously reported.
Experimental
Chemicals
Boldenone, methandrostenolone, trenbolone, testosterone,
normethandrolone, nandrolone, mibolerone, and
methenolone (Scheme 1, where the numbers in boldenone
structure show the n (...truncated)