Ionization and collision induced dissociation of steroid bisglucuronides

Studies on steroid metabolism are of utmost importance to improve the detection capabilities of anabolic androgenic steroids (AASs) misuse in sports drug testing. In humans, glucuronoconjugates are the most abundant phase II metabolites of AAS. Bisglucuronidation is a reaction where two separated functional groups on the same molecule are conjugated with glucuronic acid. These metabolites have not been studied in depth for steroids and could be interesting markers for doping control. The aim of the present work was to study the ionization and collision‐induced dissociation of steroid bisglucuronides to be able to develop mass spectrometric analytical strategies for their detection in urine samples after AAS administration. Because steroid bisglucuronides are not commercially available, 19 of them were qualitatively synthesized to study their mass spectrometric behavior. Bisglucuronides ionized as [M+NH₄] ⁺ in positive mode, and as [M−H]⁻ and [M−2H] ²⁻ in negative mode. The most specific product ions of steroid bisglucuronides in positive mode resulted from the neutral losses of 387 and 405 Da (corresponding to [M+NH₄₋ NH₃−2gluc−H₂O] ⁺ and [M+NH₄−NH₃₋2gluc−2H₂O] ⁺, respectively, being “gluc” a dehydrated glucuronide moiety), and in negative mode, the fragmentation of [M−2H] ²⁻ showed ion losses of m/z 175 and 75 (gluc⁻ and HOCH₂CO₂ ⁻, respectively). On the basis of the common behavior, a selected reaction monitoring method was developed to detect bisglucuronide metabolites in urine samples. As a proof of concept, urines obtained after administration of norandrostenediol were studied, and a bisglucuronide metabolite was detected in those urines. The results demonstrate the usefulness of the analytical strategy to detect bisglucuronide metabolites in urine samples, and the formation of these metabolites after administration of AAS.