Detection and characterization of anabolic steroids in doping analysis by LC-MS

The detection of target anabolic steroids in doping analysis has generally been performed by gas chromatography combined with mass spectrometry (GC-MS). However, liquid chromatography combined with tandem mass spectrometry (LC-MS2) is gradually becoming more important for this purpose. Also, since non-commercially available anabolic steroids have been found in some doping-control samples, detection and structural determination of unknown steroids has become a challenge for doping-control laboratories. We discuss the potential of different LC-MS and LC-MS2 scan modes for detection of both target and unknown anabolic steroids. Several modes (e.g., selected reaction monitoring, full scan and product-ion scan) can be successfully used for the detection of target analytes. In order to detect and to characterize unknown steroids and metabolites, the most powerful approach seems to be to combine several scan modes. We use a practical case to illustrate the potential of LC-MS and LC-MS2 for this purpose. © 2008 Elsevier Ltd. All rights reserved

Direct quantification of steroid glucuronides in human urine by liquid chromatography-electrospray tandem mass spectrometry

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantification of glucuronides of testosterone (TG), epitestosterone (EPG), androsterone (AG) and etiocholanolone (ETG) has been developed. The method allowed for the direct determination of these analytes avoiding hydrolysis and derivatization, which are usual steps in commonly used methods based on gas chromatography-mass spectrometry (GC-MS). The electrospray ionization and the product ion spectra of the glucuronides have been studied in order to obtain the most specific transitions. The use of the selected transitions is necessary for the determination of the analytes at low ng/ml concentration levels. Two different approaches have been tested for sample preparation: direct injection after filtration and acidic liquid-liquid extraction (LLE) with ethyl acetate. Both approaches have been validated obtaining satisfactory values for accuracy and precision with limits of detection lower than 1 ng/ml for TG and EPG. Ion suppression was more pronounced after LLE probably due to the concentration of interferences from acidic urine. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC-MS method. Results have shown a good correlation between both methods with correlation coefficients higher than 0.97. A slope close to 1 was obtained for all analytes except for AG possibly due to losses during the extraction process prior to GC-MS. © 2008 Elsevier B.V. All rights reserved

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC-MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC-QTOF MS. © 2009 Elsevier Inc. All rights reserved