Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Optimisation and validation of a new analytical method for the determination of four natural and synthetic hormones using LC-ESI-MS/MS.

A rapid liquid chromatographic-tandem mass spectrometric method was developed for the simultaneous determination of four natural and synthetic hormone residues (progesterone, testosterone, trenbolone acetate and zeranol) in animal tissue samples. Sample preparation was optimised to minimise time and solvent consumption. Meat samples were mechanically homogenised and digested in a procedure that gave similar recoveries to those enzymatically hydrolysed by Helix pomatia. Efficient extraction was achieved using acidified acetonitrile (1% acetic acid). Chromatographic conditions were optimised to minimise matrix effects. Analytes were separated using a C18 column with gradient elution using ammonium formate solution in methanol (MeOH)/water (1:9) and MeOH mobile phases. Finally, residues were qualitatively and quantitatively determined by electrospray ionisation tandem mass spectrometry in multiple reaction monitoring mode. Different parameters for LC-MS/MS (e.g., declustering potential and collision energy) were optimised using API 6500QT; all analytes were measured using positive-mode electrospray ionisation (ESI+) except zeranol which was measured in negative mode (ESI-). Due to LC-MS/MS signal enhancement/suppression, the determination of hormones was based on matrix-matched standard calculations. The method was validated for the four hormones on meat samples at different fortification levels and showed accepted performance criteria according to European Commission Decision 2002/657/EC. Decision limits and detection capabilities were estimated for all analytes.

Radiolabelling of steroids: the synthesis of 17α-[4-(14) C]trenbolone.

17ß-Hydroxyestra-4,9,11-trien-3-one or trenbolone is an anabolic steroid used in some meat producing countries where its use is licenced. In cattle it is metabolised into 17α-trenbolone. We were required to make 17α-[4-(14) C]trenbolone for use in environmental fate studies. At the same time we also had a request to make 17α-[4-(14) C]estradiol so we combined the two syntheses and made use of the synergy to allow us to make a batch of 17α-[4-(14) C]estradiol by known methodology and then elaborate a portion of this into 17α-[4-(14) C]trenbolone. The synthesis of 17α-[4-(14) C]trenbolone from 17α-[4-(14) C]estradiol was achieved in 8 steps and 3.1% overall yield to give material with a radiochemical purity of 99.5% and specific activity of 59 mCi/mmol.