Anabolic steroid screening analysis in urine is complex and labor intensive requiring sensitive instrumentation and optimized chromatographic separations. This research presents the practical application of comprehensive two-dimensional gas chromatography–time-of-flight-mass spectrometry (GC×GC–TOF-MS) for the identification and quantification of five androgenic anabolic steroids in urine. Conventional methods for steroid analysis in urine rely heavily on one-dimensional GC separations and selected ion monitoring (SIM) MS methods. This study utilizes GC×GC to increase peak capacity and resolution in combination with time-of-flight mass spectrometry (TOF-MS) detection followed by data processing with deconvolution software algorithms for positive confirmation of anabolic steroids in urine.

A steroid mixture containing stanozolol (Winstrol), 4-hydroxystanozolol, boldenone, 19-norandrosterone, 17α-methylandrostan-3α-17β -diol, and 3-hydroxystanozolol was prepared from commercial standards. Experimental results for the identification of 3-hydroxystanozolol at the 2-ppb level are presented. The illegal anabolic steroid stanozolol and metabolites are known to be particularly difficult to detect and separate chromatographically. Methyltestosterone was used as an internal standard (ISTD). Stanozolol was not included in the calibration curve development due to poor chromatographic response below 10 ppb. Although urine sampling is simple and easy to obtain, it poses a variety of challenges for the laboratory analyst. Sample preparation is labor intensive, complex, and difficult to reproduce consistently. Urine is a complicated biochemical mixture. The matrix effects from urine often can obscure detection of trace-level steroids and their metabolites. Instrumentation must be able to provide absolute confirmation of the analytical results. Sample preparation followed a well established extraction and derivatization procedure for antidoping control. Results from this study show significant improvements in chromatographic resolution and peak capacity, as well as the enhanced detectability that GC×GC–TOF-MS provides for antidoping control screening. Successful trace level identifications of the five steroid standards mixture will be shown at the 2-ng/mL (2-ppb) level. This exploratory research investigation demonstrates favorable and practical applicability of GC×GC–TOF-MS for the positive identification of anabolic steroids at the lowest allowable concentration limits which meet the strict guidelines set by the World Anti-Doping Agency (WADA). The increased peak capacity and enhanced chromatographic resolution of GC×GC coupled with a fast acquisition TOF-MS, up to 500 Hz, is essential for the successful acquisition and analysis of the data density needed to characterize low levels of steroids fully in complex sample matrices such as urine. These data-rich files are processed with deconvolution algorithms, which deliver qualitative identification as well as a multiple compound quantification in a single run. The results show limit-of-detection values at or below 2 ppb for five anabolic steroids with a calibration linearity of greater than 99.9%.