Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

• LC–HRMS screening method for the detection of a variety of anabolics in hair.
• Detection of unmetabolized anabolic steroids and their esters in hair matrix by simple keratin pretreatment.
• Identification of target compounds by retention time, accurate mass and isotopic cluster.
• Quantitative determination of detected compounds.
• Possibility to a retrospective re-analysis of the acquired datafile in case a further analyte is to be screened.

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.The limits of detection obtained varied from 10 to 50 pg mg−1, and limits of quantitation were 0.5 ng mg−1 for all compounds. The method was linear for all analytes in the ranges from the LOQ to 6 ng mg−1, giving correlation coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Specificity was assessed by analysing ten blank samples and fifteen samples from polidrug abusers. This method was applied to a real-life case, resulting in the identification of testosterone undecanoate in the hair of a suspect. The analyte identity was confirmed by the analysis of its in-source fragmentation and comparison to a certified standard. Thanks to the scan acquisition, this method also enables retrospective re-analysis of the acquired datafile in case a further analyte needs to be screened.

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