Two-dimensional gas chromatography with heart-cutting for isotope ratio mass spectrometry analysis of steroids in doping control

The accuracy and precision of gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) measurements are highly dependent on analyte purity. Reliable analysis of urinary steroids for doping control therefore requires extensive and time‐consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC‐C‐IRMS analysis. The use of two‐dimensional GC (GC‐GC) with heart‐cutting (Deans Switch) as a possible approach to reduce the sample purification required for IRMS analysis is described herein. The system uses a low thermal mass oven (LTM) incorporated into an existing GC‐C‐IRMS system. GC‐GC allowed the use of a cyanopropyl/phenyl column in the first dimension to optimize the separation of underivatized steroids, while a phenyl‐methylpolysiloxane column in the second dimension focuses the selectively cut analytes into narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte identity, eluent from the second GC was split, with 20 % entering a scanning MS, and 80 % flowing to the IRMS. As a proof concept, the developed method was then used to analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of Testogel®). The T delta value (−27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11‐ketoetiocholanolone (−22.5 ‰) (used as an endogenous reference compound (ERC)), indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the peak to be T. Copyright © 2012 John Wiley & Sons, Ltd. Two‐dimensional GC (GC‐GC) with heart‐cutting is investigated as an approach to simplify sample purification for the IRMS analysis of underivatised urinary steroids. A cyanopropyl/phenyl column was used in the GC1 to optimise steroid separation, before a phenyl‐methylpolysiloxane column focused analytes into narrower peaks prior to MS analysis. In addition, eluent from GC2 was split, with> 80 % flowing to the IRMS and the remainder entering a scanning MS facilitating peak identification.