Development of an analytical method for the determination of the misuse in sports of boldenone through the analysis of urine by on-line coupling liquid chromatography–gas chromatography–combustion–isotope ratio mass spectrometry

Abstract

Boldenone (Bo), androsta-1,4-dien-17β-ol-3-one, is an anabolic androgenic steroid not clinically approved for human application. Despite this, many cases are reported every year of athletes testing positive for Bo or its main metabolite 5β-androst-1-en-17β-ol-3-one (BoM). Recently the capability of different human intestinal bacteria to produce enzymes able to modify endogenous steroids in Bo has been demonstrated. When a urinary concentration of Bo and/or BoM between 5 and 30ng/mL is measured a complementary analysis by gas chromatography combustion isotope ratio mass spectrometry (GC–C–IRMS) must be carried out to discriminate the endogenous or exogenous origin. In the present work, a novel analytical method that couples LC–GC by means of the TOTAD interface with C–IRMS is described. The method is based on a first RPLC separation of unacetyled steroids, followed by acetylation and automated on-line LC–GC–C–IRMS, which includes a second RPLC clean-up of acetyl Bo and BoM, isolation of the two fractions in a fraction collector and their consecutive analysis by GC–C–IRMS. The method has been applied to the analysis of urine samples fortified at 5 and 10ng/mL, where it has shown a good performance.