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

•A new analytical method of boldenone and its main metabolite in urine is presented.•On-line coupling LC–GC–C–IRMS is presented for the first time.•The method has been applied to the analysis of fortified urine samples.•The method shows good repeatability and very good sensitivity.•The high sensitivity opens the possibility of decreasing the urine sample volume. 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.