In this paper, the use of ferrocene-based "electrochemically ionizable" derivatives to enhance ES-MS analysis of simple alcohols, sterols, and phenols is discussed. These derivatives are designed to take advantage of the electrolysis process inherent to operation of the ES ion source for selective ionization. Derivatization procedures, electrochemical character of the derivatives, and the ES-MS operational parameters necessary to maximize electrochemical ionization and to enhance gas-phase detection are presented with reference to ferrocenecarbamate ester derivatives of a variety of alcohol standards, as well as the ferroceneboronate derivative of the diol, pinacol. Tandem mass spectrometric analysis of the derivatives (precursor and product ion spectra) is shown to provide derivative confirmation, enhanced detection, and additional analyte structure information. The utility of this derivatization approach for the selective detection of alcohols in complicated mixtures is demonstrated using a saw palmetto (Serenoa repens) fruit extract known to contain a variety of alcohols at low levels.
We herein report upon an approach whereby the interpretation of tandem mass spectrometry spectra can be both expedited and simplified via the accurate mass assignment of product ions utilizing a tandem quadrupole time-of-flight mass spectrometer (QqTOF). The applicability of the QqTOF in the drug metabolism laboratory is illustrated by the elucidation and differentiation of the dissociative pathways for Bosentan and its hydroxylated and demethylated metabolites. Target analyte fragmentation mechanisms were readily achieved by the measurement of product ions with a mass accuracy <5 ppm, possible by single-point internal recalibration using the residual precursor ion as calibrant. Differentiation of both precursor and product ions from nominally isobaric matrix species derived from biological extracts is demonstrated by operation of the QqTOF at resolutions of not, vert, similar8000 (m/ΔmFWHM)
The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M ϩ 2Na] ϩ· radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions. [3,4], the analysis of lipids using tandem mass spectrometry (MS) has been greatly facilitated since these techniques can generate the intact gaseous pseudo-molecular ions for most lipids ranging from simple fatty acids to complex lipids [5][6][7][8][9][10]. Among the various complex lipids, glycerophospholipids (GPLs) perform two important biological functions: one is making up most of the membranes of mammalian cells, and the other is acting as secondary messengers in metabolism [11,12]. The structural determination (identities and positions of the fatty acid substituents) of GPLs for all five subclasses has been made via CID of negative ions formed by either ESI, fast atom bombardment (FAB), or MALDI [5,13,14]. Among the five main subclasses of GPLs, which are differentiated by the head-group (e.g., ethanolamine, choline, serine, glycerol, or inositol), glycerophosphocholine (GPC) species generally give stronger signals in positive ion mode than in negative mode due to the fixed charge on the quaternary nitrogen. The structural information mentioned above can be obtained by CID of alkali adducts of GPC species generated in positive ion ESI-MS [15]. Little or no information regarding the location of the double bonds in GPCs has been reported by these methods.Both electron capture dissociation (ECD) [16 -19] and electron-transfer dissociation (ETD) [20 -23] have been shown to be particularly useful in the structural determination of proteins and peptides [24 -27]. This is mainly because more extensive sequence information can often be obtained via ECD or ETD than via CID and because, unlike CID, labile post-translational modifications are often retained, which is particularly va...
Neonatal plasma contains interferents that increase the apparent 17 alpha-hydroxyprogesterone (17-OHP) content measured by direct (no-extraction) radioimmunoassay. We fractionated extracts from neonatal plasma pools by liquid chromatography with a Sephadex LH-20 column and measured 17-OHP immunoreactivity by a direct test kit. We found immunoreactivity in the free steroid and glucuronide fraction and also in the monosulfate fraction. We analyzed these two fractions by reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. We collected fractions and assayed for 17-OHP immunoreactivity. The HPLC fractions containing the interfering steroid monosulfates were analyzed by ion-spray mass spectrometry and, after solvolysis, by gas chromatography-mass spectrometry. Several monosulfates were identified, including those of 17 alpha-hydroxy-pregnenolone, 16 alpha-hydroxypregnenolone, pregnenolone, and several pregnenetriols. 17 alpha-Hydroxypregnenolone sulfate was the most significant interferent. Other commercially available 17-OHP assays showed similar interference when used without an extraction step. Kit manufacturers should select antibodies and protocols to minimize cross-reaction with sulfates, especially 17 alpha-hydroxypregnenolone sulfate.
Frontal affinity chromatography (FAC) interfaced with electrospray mass spectrometry (ESI-MS) has been reported as a potential method for screening of compound mixtures against immobilized target proteins. However, the interfacing of bioaffinity columns to ESI-MS requires that the eluent that passes through the protein-loaded column have a relatively low ionic strength to produce a stable spray. Such low ionic strength solvents can cause serious problems with protein stability and may also affect binding constants and lead to high nonspecific binding to the column. Herein, we report on the interfacing of bioaffinity columns to matrix-assisted laser desorption/ionization (MALDI) MS/MS as a new platform for FAC/MS studies. Capillary columns containing a monolithic silica material with entrapped dihydrofolate reductase were used for frontal affinity chromatography of small-molecule mixtures. The output from the column was combined with a second stream containing alpha-cyano-hydoxycinnamic acid in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer-controlled x-y stage, creating a semipermanent record of the FAC run. The use of MALDI MS/MS allowed for buffers with significantly higher ionic strength to be used for FAC studies, which reduced nonspecific binding of ionic compounds and allowed for better retention of protein activity over multiple runs. Following deposition, MALDI analysis required only a fraction of the chromatographic run time, and the deposited track could be rerun multiple times to optimize ionization parameters and allow signal averaging to improve the signal-to-noise ratio. Furthermore, high levels of potential inhibitors could be detected via MALDI with limited ion suppression effects. Both MALDI- and ESI-based analysis showed similar retention of inhibitors present in compound mixtures when using identical ionic strength conditions. The results show that FAC/MALDI-MS should provide advantages over FAC/ESI-MS for high-throughput screening of compound mixtures.
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