The use of Anabolic Androgenic Steroids (AAS) to enhance performance has been a concern for decades in both human and animal sports. Complicating the matter is that some of these agents can be endogenously produced at various levels in the horse and regulatory authorities, such as the Horseracing Integrity and Safety Authority (HISA), have regulations to address their abuse. The current methods of AAS detection and quantitation primarily use Gas Chromatography-Mass Spectrometry (GC-MS) or Liquid Chromatography-Mass Spectrometry (LC-MS) using urine, serum/plasma, and hair samples. These approaches have some limitations, including time-consuming sample preparation with cleavage of glucuronide and sulfated metabolites, lack of reference material for the phase II metabolites, and the need to derivatize some AAS for adequate detection using mass spectrometry. This project concentrated on the comparison of different deconjugation approaches for sulfated and glucuronidated AAS metabolites to their corresponding free drug. The following metabolites were tested: testosterone sulfate, epitestosterone sulfate, nandrolone sulfate, boldenone sulfate, testosterone glucuronide, epitestosterone glucuronide, nandrolone glucuronide, epinandrolone glucuronide, and boldenone glucuronide. Phase II metabolites were deconjugated using enzymatic hydrolysis, using sulfatases and b-glucuronidases from three different manufacturers, or chemical hydrolysis, including methanolysis and solvolysis. The deconjugation approaches were compared utilizing a protocol that included a weak anion exchange solid-phase extraction followed by the different enzymatic and chemical hydrolyses protocols. Following the deconjugation approaches, the samples were subjected to liquid-liquid extraction (LLE), and a solid-phase extraction (SPE) using an amine cartridge prior to liquid chromatography-mass spectrometry (LC-MS) analysis. Samples were compared at different incubation times with and without matrix. Comparing all deconjugation methods, the use of both the Integrated Micro-Chromatography Systems’ (IMCS’) sulfatase A and β-glucuronidase enzymes would allow for an acceptable and reliable approach to cleave phase II AAS metabolites for a screening protocol
