Improved Steroids Detection and Evidence for Their Regiospecific Decompositions Using Anion Attachment Mass Spectrometry.

Nonpolar anabolic steroids are doping agents that typically do not provide strong signals by electrospray ionization-mass spectrometry (ESI-MS) owing especially to the low polarity of the functional groups present. We have investigated the addition of anions, in ammonium salt form, to anabolic steroid samples as ionization enhancers and have confirmed that lower instrumental limits of detection (as low as 10 ng/mL for fluoxymesterone-M) are obtained by fluoride anion attachment mass spectrometry, as compared to ESI(+)/(-) or atmospheric pressure photoionization (APPI)(+). Moreover, collision-induced decomposition (CID) spectra of precursor fluoride adducts of the bifunctional steroid "reduced pregnenolone" (containing two hydroxyl groups) and its d4-analogue provide evidence of regiospecific decompositions after attachment of fluoride anion to a specific hydroxyl group of the steroid. This type of charting of specific CID reaction pathways can offer value to selected reaction monitoring experiments (SRM) as it may result in a gain in selectivity in detection as well as in improvements in quantification.

Novel fragmentation pathways of anionic adducts of steroids formed by electrospray anion attachment involving regioselective attachment, regiospecific decompositions, charge-induced pathways, and ion-dipole complex intermediates.

The analysis of several bifunctional neutral steroids, 5-α-pregnane diol (5-α-pregnane-3α-20ßdiol), estradiol (3,17α-dihydroxy-1,3,5(10)-estratriene), progesterone (4-pregnene-3,20-dione), lupeol (3ß-hydroxy-20(29)-lupene), pregnenolone (5-pregnen-3ß-ol-20-one), and pregnenolone acetate (5-pregnen-3ß-ol-20-one acetate) was accomplished by negative ion electrospray mass spectrometry (ESI-MS) employing adduct formation with various anions: fluoride, bicarbonate, acetate, and chloride. Fluoride yielded higher abundances of anionic adducts and more substantial abundances of deprotonated molecules compared with other investigated anions. Collision-induced dissociation (CID) of precursor [M + anion](-) adducts of these steroids revealed that fluoride adduct [M + F](-) precursors first lose HF to produce [M - H](-) and then undergo consecutive decompositions to yield higher abundances of structurally-informative product ions than the other tested anions. In addition to charge-remote fragmentations, the majority of CID pathways of estradiol are deduced to occur via charge-induced fragmentation. Most interestingly, certain anions exhibit preferential attachment to a specific site on these bifunctional steroid molecules, which we are calling "regioselective anion attachment." Regioselective anion attachment is evidenced by subsequent regiospecific decomposition. Regioselective attachment of fluoride (and acetate) anions to low (and moderate) acidity functional groups of pregnenolone, respectively, is demonstrated using deuterated compounds. Moreover, the formation of unique intermediate ion-dipole complexes leading to novel fragmentation pathways of fluoride adducts of pregnenolone acetate, and bicarbonate adducts of d(4)-pregnenolone, are also discussed.

Characterization of an exception to the 'even-electron rule' upon low-energy collision induced decomposition in negative ion electrospray tandem mass spectrometry.

Electrospray-generated precursor ions usually follow the 'even-electron rule' and yield 'closed shell' fragment ions. We characterize an exception to the 'even-electron rule.' In negative ion electrospray mass spectrometry (ES-MS), 2-(ethoxymethoxy)-3-hydroxyphenol (2-hydroxyl protected pyrogallol) easily formed a deprotonated molecular ion (M-H)(-) at m/z 183. Upon low-energy collision induced decomposition (CID), the m/z 183 precursor yielded a radical ion at m/z 124 as the base peak. The radical anion at m/z 124 was still the major fragment at all tested collision energies between 0 and 50 eV (E(lab)). Supported by computational studies, the appearance of the radical anion at m/z 124 as the major product ion can be attributed to the combination of a low reverse activation barrier and resonance stabilization of the product ions. Furthermore, our data lead to the proposal of a novel alternative radical formation pathway in the protection group removal of pyrogallol.

Unusual odd-electron fragments from even-electron protonated prodiginine precursors using positive-ion electrospray tandem mass spectrometry.

Reports of anticancer and immunosuppressive properties have spurred recent interest in the bacterially produced prodiginines. We use electrospray tandem mass spectrometry (ES-MS/MS) to investigate prodigiosin, undecylprodiginine, and streptorubin B (butyl-meta-cycloheptylprodiginine) and to explore their fragmentation pathways to explain the unusual methyl radical loss and consecutive fragment ions that dominate low-energy collision-induced dissociation (CID) mass spectra. The competition between the formation of even-electron ions and radical ions is examined in detail. Theoretical calculations are used to optimize the structures and calculate the energies of both reactants and products using the Gaussian 03 program. Results indicate that protonation occurs on the nitrogen atom that initially held no hydrogen, thus allowing formation of a pseudo-seven-membered ring that constitutes the most stable ground state [M + H](+) structure. From this precursor, experimental data show that methyl radical loss has the lowest apparent threshold but, alternatively, even-electron fragment ions can be formed by loss of a methanol molecule. Computational modeling indicates that methyl radical loss is the more endothermic process in this competition, but the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. Additionally, this characteristic and unusual loss of methyl radical (in combination with weaker methanol loss) from each prodiginine is useful for performing constant neutral loss scans to quickly and efficiently identify all prodiginines in a complex biological mixture without any clean-up or purification. The feasibility of this approach has been proven through the identification of a new, low-abundance prodigiosin analog arising from Hahella chejuensis.