Profiling of 19-norsteroid sulfoconjugates in human urine by liquid chromatography mass spectrometry

Abstract

19-Nortestosterone (nandrolone) major metabolites in human urine are excreted as sulfoconjugated and glucuroconjugated forms. A sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in negative ESI mode was developed for direct quantification of 19-norandrosterone sulfate (19-NAS) and 19-noretiocholanolone sulfate (19-NES). For both sulfoconjugates, the [M−H] − ion at m/ z 355 and the fragment ion at m/ z 97 were used as the precursor and product ions, respectively. The purification method involved a complete and rapid separation of sulfates and glucuronides in two extracts after loading the sample on a weak anion exchange solid phase extraction support (SPE Oasis ® WAX). Then, sulfates were separated by LC (Uptisphere ® ODB, 150 mm × 3.0 mm, 5 μm) and analyzed on a linear trap and a triple quadrupole mass spectrometer. The lower limit of detection (LLOD) and lowest limit of quantification (LLOQ) were of 100 pg mL −1 and 1 ng mL −1, respectively. Assay validation demonstrated good performances in terms of trueness (92.0–104.9%), repeatability (0.6–7.2%) and intermediate precision (1.3–10.8%) over the range of 1–2500 ng mL −1. Finally, 19-NAS and 19-NES in urine samples collected after intake of 19-norandrostenedione (nandrolone precursor) were quantified. This assay may be easily implemented to separate glucuronide and sulfate steroids from urine specimens prior to quantification by LC/MS/MS.