Mass spectral fragmentation analyses of isotopically labelled hydroxy steroids using gas chromatography/electron ionization low‐resolution mass spectrometry: A practical approach

Abstract

Rationale:Gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) is used for routine screening of anabolic steroids in many laboratories after the conversion of polar groups into trimethylsilyl (TMS) derivatives. The aim of this work is to elucidate the origin and formation of common and subclass-specific fragments in the mass spectra of TMS-derivatized steroids. Especially in the context of metabolite identification or analysis of designer drugs, isotopic labelling is helpful to better understand fragment ion generation, identify unknown compounds and update established screening methods. Methods: Stable isotope labelling procedures for the introduction of [ 2 H 9 ]-TMS or 18 O were established to generate perdeuterotrimethylsilylated, mixed deuterated and 18 O-labelled derivatives for 13 different hydroxy steroids. Fragmentation proposals were substantiated by comparison of the abundances of isotopically labelled and unlabelled fragment ions in unit mass resolution GC/MS. Specific fragmentations were also investigated by high-resolution MS (GC/quadrupole time-of-flight MS, GC/QTOFMS). Results: Methyl radical cleavage occurs primarily from the TMS groups in saturated androstanes and from the steroid nucleus in the case of enol-TMS of oxo or α,β-unsaturated steroid ketones. Loss of trimethylsilanol (TMSOH) is dependent on steric factors, degree of saturation of the steroid backbone and the availability of a hydrogen atom and TMSO group in the 1,3-diaxial position. For the formation of the [M -105] + fragment ion, methyl radical cleavage predominates from the angular methyl groups in position C-18 or C-19 and is independent of the site of TMSOH loss. The common [M -15 -76] + fragment ion was found in low abundance and identified as [M -CH 3 -(CH 3 ) 2 SiH -OH] + . For the different steroid subclasses further diagnostic fragment ions were discussed and structure proposals postulated.Conclusions: Stable isotope labelling of oxo groups as well as derivatization with deuterated TMS groups enables the detection of structure-related fragment ion

In‐depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3‐keto‐4‐ene hydroxy steroids

“…In the mass spectra of all perdeuterotrimethylsilylated and mixed deuterated standards ( #.2B , #.3B , #.4B , Tables 1 and 2) mainly [M – CH 3 ] + = [M – 15] + fragment ions were observed and only a very low abundance of [M – CD 3 ] + = [M – 18] + ions. This finding was confirmed with MS/MS experiments ([M] •+ as precursor) and complies with the assumption that in enol‐TMS and Δ4 steroids methyl radical loss originates predominantly from the steroid nucleus as reported in the literature 12,32 …”

“…Suitable hydrogen atoms are located in the 1,3‐diaxial position from the TMS oxygen and the bonding distance needs to be less than 3.2 Å 35 ( #.1 , Tables 3 and 4). The dependence of TMSOH loss on hydrogen availability and bonding distance was recently demonstrated via 18 O‐labelling for different hydroxy steroids, for example 5α‐ and 5β‐androstanediols 12 …”

“…To obtain 4‐hydroxyandrost‐4‐ene‐3,17‐[ 18 O 2 ]‐dione ([ 18 O 2 ]‐formestane, 3 ), the two oxo functions of formestane ( 2 ) were isotopically labelled utilizing H 2 18 O via an imine intermediate as previously described 11,12 . Formestane (1.5 mg) was dissolved in toluene (900 μL).…”

“…Individual stock solutions (1 mg/mL) of compounds 1 , 2 and 4 – 10 were prepared in ACN and stored at −18°C prior to GC/MS analysis. Aliquots of 10 μL of each stock solution were evaporated at 100°C and the respective residues derivatized with either MSTFA or [ 2 H 18 ]‐BSA, or both, using four different derivatization protocols 12 : pertrimethylsilylation: 90 μL MSTFA/TMIS reagent (MSTFA/NH 4 I/ethanethiol, 1,000:2:3, v/w/v) at 60°C for 15 min (to obtain derivatives #.1 , Tables 3 and 4) perdeuterotrimethylsilylation: 80 μL [ 2 H 18 ]‐BSA/TMIS reagent (5 μL of a saturated NH 4 I solution in 2‐mercaptoethanol added to 100 μL [ 2 H 18 ]‐BSA) at 90°C for 30 min (to obtain derivatives #.2 , Tables 3 and 4) mixed deuterated derivatives: …”

Response to “Molecular ion: A more contemporary definition”

“…Many practitioners of mass spectrometry do not have access to instrumentation capable of accurate mass measurement, and numerous applications of nominal m/z measurement, where the m/z of the ion and mass of the molecule are the same numerical value, still exist. [3][4][5][6][7][8][9][10][11][12][13] Therefore, the advancements in instrument capability are not universally available and do not justify amending the definition of "molecular ion".…”