Subsequent GC-MS and NMR investigations identified a mixture of 3- and 3- isomers of the new compound; 3-chloro-17-methyl-5-. Because of the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3-chloro-17-methyl-5-androstan-17-ol were performed using equine and human S9 liver microsomal fractions. The subsequent discovery of deoxymethyltestosterone (17-methyl-5-androst-2-en-17-ol; . DMT or Madol)[12] soon after prompting anti-doping laboratories to perform additional.
We describe herein the identification, synthesis, in vitro androgenic bioactivity, in vitro metabolic studies using both equine and human liver S9 fraction, and gas chromatography-tandem mass spectrometry (GC-MS-MS) methods for detection in routine anti-doping analysis of 3-chloro-17-methyl-5-androstan-17-ol. The residue was taken up in 1 ml of the eluent solution (10% ethyl acetate/n-hexane) and loaded onto a silica gel (Kieselgel mm, Merck) column of approx. Synthesis of 17-methyl-5-androstane-317-diol and 17-methyl-5-androstane-317-diol used in this study was previously described by McKinney et al.[ 22] Melting points were determined using an SRS Optimelt MPA 100 melting point apparatus and are uncorrected.
They can be obtained by methylation of the corresponding known 3-chloro-5α-androstan-17-ones[23] which in turn can be synthesized from 3-hydroxy-5α-androstan-17-ones by chlorination with inversion of configuration in reaction centers.[24] For this purpose, epiandrosterone (1) was subjected to chlorination to provide 3α-chloro-5α-androstan-17-one (2)[23] in 79% yield (Figure 1). Formation of the enol acetate (7) from the ketone (2) followed by a sequence involving epoxidation, rearrangement and acetylation gave the acetoxy-ketone (8). Cells were then treated with steroids (5 µL/well) at a concentration range to generate a sigmoidal dose curve used to calculate the EC50 of the steroid response.
The resulting supernatant solution was decanted and extracted with ethyl acetate (2 x 1 ml) and the organic layer was dried under a stream of nitrogen. Residues were analyzed by GC-MS-MS after forming 1.) TMS derivatives as described above and 2.) acetonide-TMS derivatives to investigate C16-C17 stereochemistry using a modified version of the procedure described by Houghton and Dumasia [ 28]. specifically, reaction of acetone (0.5 mL) and trifluoroacetic acid (50 L) at 55 °C for 60 min, drying under N2 at 60 °C, followed by TMS-ether formation.
Results
The 400 MHz 1H NMR spectrum of the mixture (Supporting Information, Figure S1) showed a high degree of complexity with significant overlap of resonances, both due to the relatively poor dispersion of the aliphatic resonances at relatively low field strength and the presence of two species in the sample. Expansions of the 400 MHz 1H NMR spectrum of the mixture focusing on resonances attributed to protons attached to the carbon bearing the chloro substituent are shown in Figure 5. NOE, 13C chemical shift and gHMBC data obtained for the mixture confirmed the attachment of the chloro substituents to C3 in both compounds.
2D gHSQC and gHMBC analysis of the sample facilitated assignment of the relevant 13C chemical shifts (Supporting information, Figures S3 and S5). Confirmation of the attachment of the chlorine substituent to the C3 carbons of the two compounds was obtained through a series of selective NOE experiments (Supporting information, Figure S6). The chemical shifts of the protons and the carbon atoms of the C and D rings of both compounds closely matched, indicating similar substitution in those rings.
Strong NOE enhancement of the resonances of both the C18 and C20 methyl groups as well as enhancement of the adjacent βC16 and equatorial C12 methylene proton resonances were observed. Production experiments targeting m/z 484 identified a response at Rt=15.46 min, indicating that the 3-chloro substituent in the parent steroid was retained. Presumptive identification of 3-chloro-17-methyl-5-androstane-16,17-diol was based on 1.) the presence of m/z 231 reported by McKinney et al.[22] and Schoene et al.[31] to be diagnostic of C16-hydroxylated metabolites of 17-methyltestosterone, and 2.) failure to form the cis-acetonide derivative supporting 16-stereochemistry.[28].
The identity of 3-chloro-17-methyl-5-androstane-16,17-diol was confirmed by comparison of the equine S9 fraction with the synthesized reference material (Figure 6). The identity of 17-methyl-5-androstane-3,17-diol was confirmed by comparison of human fraction S9 with a certified reference material and verification of C3 stereochemistry compared to 17-methyl-5- androstane-3,17-diol (Figure 7). The limit of detection (LOD) for each steroid was determined by triplicate analysis of three authentic equine and human blank urine samples along with steroid spikes (n=3) of the same urine samples at concentrations of 10 ng/mL.
Extraction recovery was assessed by comparing 1 and 10 ng/mL pre- and post-extraction drops in each of three equine and human blank urine samples. The estimated LOD of 0.5 ng/mL for the most sensitive target analyte transitions provides effective routine screening capability in equine urine. In addition to the metabolic and translational foci of the present study, the opportunity was taken to evaluate the application of this detection method to designer steroids in horses.
Discussion
Discrete equine and human metabolism in vitro will require further in vivo research to determine toxicity of 3-chloro-17-methyl-5-. Indeed, limitations must be considered in assuming correlation between in vitro and in vivo experiments.[36] For example, additional hydroxylation may be expected in vivo, which may provide longer detection periods. In addition, the in vivo elimination of the parent steroid cannot be neglected, so it has been included in the routine screening method.
Although there are advantages to adopting precursor ion strategies for complementary non-targeted detection of “designer” steroids, in-matrix validation is imperative to assess performance. The results of this study have highlighted the limitation in sensitivity of precursor ion scanning. Greater sensitivity can be achieved in complex matrices such as equine urine using more conventional selected ion monitoring (SIM) strategies where the MS cycle time allows.
In addition to hardware considerations, the use of differential and in-silico software applications is likely to allow anti-doping laboratories for de novo identification of.
Conclusion
Acknowledgements
Discovery, biosynthesis and structure elucidation of metabolites of a doping agent and a direct analog, tetrahydrogestrinone and gestrinone, using human hepatocytes. Detection of some designer steroids in horse urine, in Proceedings of the 16th International Conference of Racing Analysts and Veterinarians. The use of in vitro technologies and high-resolution/accurate mass LC-MS to screen for.
Current status and bioanalytical challenges in the detection of unknown anabolic androgenic steroids in doping control analysis. 25] Crystallographic data (without structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Center as Supplementary Publication no. Evaluation of the androgenic activity of nutraceutical-derived steroids using in vitro mammalian and yeast androgen bioassays.
Studies on the metabolism of anabolic steroids in the horse: the identification of some 16-oxygenated metabolites of 19-nortestosterone. Method for selective introduction of trimethylsilyl and perdeuterotrimethylsilyl groups into hydroxyl steroids and its utility in mass spectrometric interpretations. The detection of abuse of androstenedione in sports: a mass spectrometry strategy to identify the metabolite 4-hydroxyandrostenedione.
The use of in vitro technologies in combination with accurate, high-resolution mass LC-MS to study drug metabolism in equine pharmacovigilance.
List of figure legends
All four observed doublet splittings are small (~4-5Hz) and consistent with the values expected for 3Jeq,ax and 3Jeq,eq couplings, placing the H3 proton in the equator. b) An expansion of the region of the same spectrum centered on δ 3.82ppm.
