Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography–tandem mass spectrometry

Abstract

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1 ′ -carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H] + signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO 2 +H] + and [M-ImCO 2 +H] + as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples. K E Y W O R D Sanabolic androgenic steroids, derivatization, doping control, imidazole carbamates, mass spectrometry

Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods

“…All samples were prepared with final concentrations of 5 μg/mL steroid, unless otherwise indicated. Derivatization was performed by modifying previous methods for Girard’s Reagent P and 1,1-carbonyldiimidazole. , Briefly, the Girard’s Reagent P reaction was performed by combining 100 μL of a 1 mg/mL (in methanol) Girard’s Reagent P solution, 250 μL of methanol (with 10% acetic acid), and 5 μL of the 1 mg/mL steroid stock solution. The mixture was sonicated at room temperature for 15 min, dried down under nitrogen at room temperature, and reconstituted in 1 mL of mobile phase A.…”

“…Derivatization by 2-hydrazinopyridine has been applied to analysis of fathead minnows and salivary samples . Fragkaki et al specifically applied imidazole carbamate derivatization to anabolic androgenic steroids in human urine . Qin et al employed a parallel derivatization strategy using both methoxyamine and dansyl chloride to respectively target carbonyl and phenolic hydroxyl groups across a range of endogenous steroids with increased sensitivity in a study of ovarian cancer samples …”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Due to the modest ionization efficiency observed for numerous AAS‐related target analytes, AAS metabolites are more commonly tested by GC‐MS/MS than LC‐MS/MS. Improved ESI capabilities can be accomplished by derivatization strategies as demonstrated by Fragkaki et al, who studied the impact of imidazole carbamate derivatization on 34 analytes (representing 23 AAS plus zeranol) by means of 1,1′‐carbonyldiimidazole (CDI) regarding their detection by LC‐HRMS/MS 52 . Urine sample preparation protocols were largely adapted from established procedures employing enzymatic hydrolysis, liquid‐liquid‐extraction (LLE), and evaporation to dryness before derivatization.…”

Liquid chromatography–mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes

“…Chemical derivatization (chemical labeling) has been widely used in order to extend the application of LC–ESI–MS (MS) in the analysis of free AAS in complex samples 35–44 . The main approach applied is the introduction of permanently charged moieties or easily ionizable groups containing tertiary nitrogen into the functional group of steroids, 45–53 thus improving their detection in positive ESI mode. Most popular strategies include the conversion of carbonyl‐groups of oxo‐steroids to hydrazones 54,55 with quaternary ammonium and pyridinium moieties or oximes 56,57 improving their ESI sensitivity, though other derivatization processes targeting the hydroxyl groups of the steroids have been also provided efficient enhancement of their ionization efficiency.…”