The potential use of complex derivatization procedures in comprehensive HPLC‐MS/MS detection of anabolic steroids

Abstract

The use of two separate derivatization procedures with the formation of oxime (hydroxyl ammonium pretreatment) and picolinoyl (mixed anhydride method) derivates of anabolic steroids following HPLC-MS/MS analysis was proposed. The main product ions of obtained derivatives for 21 anabolic steroids were evaluated and fragmentation pathways were compared.The analysis of MS/MS spectra for underivatized steroids versus oxime or picolinoyl derivatives showed that in case of analytes containing conjugated double bonds in sterane core all of the observed MS/MS spectra contained abundant product ions of diagnostic value. The implementation of derivatization procedures to such compounds is useful for upgrading sensitivity or selectivity of the evaluated method. On the other hand, MS/MS spectra of underivatized and oxime analytes without conjugated double bonds in sterane core produce spectra with large amounts of low abundant product ions. Picolinoyl derivatives formation leads to highly specific spectra with product ions of diagnostic value coupled with sensitive and selective analysis at the same time. The intra- and inter-group comparison analysis revealed that fragmentation pathways for underivatized steroids and correspondent oxime derivatives are similar.The obtained oxime and picolinoyl derivatives provided 10-15 times higher ESI response in the HPLC-ESI-MS-selected reaction monitoring (SRM) when compared to those of underivatized molecules in positive HPLC-ESI-MS mode.Due to the laborious sample preparation we suggest to use the performed strategy for confirmation analysis purposes, metabolic studies or while the identification of new steroids or steroid-like substances.

A review of the analytical techniques for the detection of anabolic–androgenic steroids within biological matrices

“…LC usually utilizes a variation of C18 (octyldecylsilane) phase columns (Andersen & Linnet, 2014; Balcells et al, 2015; Baranov et al, 2010; Deshmukh et al, 2010; Görgens et al, 2016; Guddat et al, 2013; Kolmonen et al, 2009; Kuuranne et al, 2003; Mazzarino et al, 2008, 2010; Moon et al, 2008; Peters et al, 2010; Pozo, Deventer, et al, 2007; Pozo, Van Eenoo, et al, 2007a; Saito et al, 2010; Thomas et al, 2010; Virus et al, 2008; Viryus et al, 2009; Vonaparti et al, 2010), at varying lengths, inner diameters and particle sizes. Some previous C8 (octylsilane) column use can also be seen in the literature for AASs analysis (Samanidou et al, 2009; Schänzer et al, 2006; Thevis et al, 2006).…”

Liquid chromatography–mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes

“…Chemical derivatization (chemical labeling) has been widely used in order to extend the application of LC–ESI–MS (MS) in the analysis of free AAS in complex samples 35–44 . The main approach applied is the introduction of permanently charged moieties or easily ionizable groups containing tertiary nitrogen into the functional group of steroids, 45–53 thus improving their detection in positive ESI mode. Most popular strategies include the conversion of carbonyl‐groups of oxo‐steroids to hydrazones 54,55 with quaternary ammonium and pyridinium moieties or oximes 56,57 improving their ESI sensitivity, though other derivatization processes targeting the hydroxyl groups of the steroids have been also provided efficient enhancement of their ionization efficiency.…”