Analytical Methods for the Detection and Quantification of Trenbolone Acetate Metabolites, Altrenogest and Related Photoproducts via Liquid Chromatography Tandem Mass Spectrometry

Abstract

Photoreaction coupled with liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) was used to develop an analytical method for the detection and quantification of trenbolone metabolites, altrenogest, and related photoproducts in water. Target parent analytes were 17α-trenbolone (17α-TBOH), 17β-trenbolone (17β-TBOH), trendione (TBO), and altrenogest (ALT); target photoproducts were the metastable 5-hydroxy- and 12-hydroxy- photoproducts of 17α/17β-TBOH and TBO, and the cyclo-addition and hydroxy-cyclo-addition photoproducts of altrenogest. Photoproducts were generated with a solar simulator reacting trenbolone metabolite (or altrenogest) samples for 6 hrs (>10 half lives). Samples were extracted with C18 solid phase extraction (SPE) cartridges before liquid chromatographic separation with a reverse-phase C18 column with water and methanol mobile phases and ESI+ MS detection. Method detection limits (MDL's) and quantification limits (MQL's) for all compounds were near or below 1 ng L-1 except for 17β-TBOH photoproducts, which had MDL's <2 ng L-1 and MQL's <4 ng L-1. Matrix suppression was <30% using lake water, and SPE recovery rates were near/above 100%, except for 17β-TBOH photoproducts, with recoveries >75%. Intra-day relative standard deviations (RSD's) were <30% for TBO and its products, <20% for all others.