The deprotonation site of p-hydroxybenzoic acid upon electrospray ionization has been a subject of fervent debate in several articles in the Journal of the American Chemical Society and elsewhere. General consensus is that electrospray ionization mass spectrometry (ESI-MS) experimental results reflect the situation in solution to a considerable extent. Our research, using ion-mobility mass spectrometry, challenges the notion that ESI-MS results directly reflect solution-phase structures and demonstrates that the relative populations of the thermodynamically less favored gaseous carboxylate tautomer or the thermodynamically more favored gaseous phenoxide tautomer, generated from the same aqueous solution of p-hydroxybenzoic acid by ESI, can be varied back and forth by changing the probe position, capillary voltage, desolvation-gas temperature, sample infusion flow rate, and cone voltage. In other words, solvent effects are not the primary criteria that determine the relative population distributions of tautomeric carboxylate (C–) and phenoxide (P–) ions (m/z 137) generated by electrospray ionization of p-hydroxybenzoic acid. In addition, we propose that the observed ratio of the P– and C– forms indirectly reflects the relative contribution of the charge-residue or ion-evaporation process that occurs during the electrospray ion generation process.

Upon activation in the gas phase, protonated alkyl dihydrocinnamates undergo an alcohol loss. However, the mechanism followed is not a simple removal of an alkanol molecule after a protonation on the alkoxy group. The mass spectrum of the m/z 166 ion for deuteron-charged methyl dihydrocinnamate showed two peaks of 1:5 intensity ratio at m/z 133 and 134 to confirm that the incipient proton is mobile. The proton initially attached to the carbonyl group migrates to the ring and randomizes before a subsequent transfer of one of the ring protons to the alkoxy group for the concomitant alcohol elimination. Moreover, protonated methyl dihydrocinnamate undergoes more than one H/D exchange. The spectra recorded from m/z 167 and 168 ions obtained for di- and tri-deuterio isotopologues showed peak pairs at m/z 134, 135 and 135, 136, at 1:2 and 1:1 intensity ratios, respectively, confirming the benzenium ion intermediate achieves complete randomization before the proton transfer. Additionally, protonated higher esters of alkyl dihydrocinnamates undergo a cleavage of the O–CH2 bond to form an ion/neutral complex, which, upon activation, dissociates generating a carbenium ion and dihydrocinnamic acid, or rearranges to generate protonated dihydrocinnamic acid and an alkene by a nonspecific proton transfer.

An enclosed atmospheric-pressure helium-plasma ionization (HePI-MS) source avoids, or minimizes, undesired back-exchange reactions usually encountered during deuterium incorporation experiments under ambient-pressure open-source conditions. A simple adaptation of an ESI source provides an economical way of conducting gas phase hydrogen/deuterium (H/D) exchange reactions (HDX) in real time without the need for complicated hardware modifications. For example, the spectrum of [2H8]toluene recorded under exposed ambient conditions showed the base peak at m/z 96 due to fast leaching of ring hydrogens because of interactions with H2O vapor present in the open source. Such D/H exchanges are rapidly reversed if the deuterium-depleted [2H8]toluene is exposed to D2O vapor. In addition to the enumeration of labile protons, our procedure enables the identification of protonation sites in molecules unambiguously, by the number of H/D exchanges observed in real time. For example, molecules such as tetrahydrofuran and pyridine protonate at the heteroatom and consequently undergo only one H/D exchange, whereas ethylbenzene, which protonates at a ring position of the aromatic ring, undergoes six H/D exchanges. In addition, carbocations generated in situ by in-source fragmentation of precursor protonated species, such as benzyl alcohol, do not undergo any rapid H/D exchanges. Because radical cations, second-generation cations (ions formed by losing a small molecule from a precursor ion), or those formed by hydride abstraction do not undergo rapid H/D exchanges, our technique provides a way to distinguish these ions from protonated molecules.

Collision-induced fragmentation of protonated N-alkyl-p-toluenesulfonamides primarily undergo either an elimination of the amine to form CH3-(C6H4)-SO2+ cation (m/z 155) or an alkene to form a cation for the protonated p-toluenesulfonamide (m/z 172). To comprehend the fragmentation pathways, several deuterated analogs of N-decyl-p-toluenesulfonamides were prepared and evaluated. Hypothetically, two mechanisms, both of which involve ion-neutral complexes, can be envisaged. In one mechanism, the S–N bond fragments to produce an intermediate [sulfonyl cation/amine] complex, which dissociates to afford the m/z 155 cation (Pathway A). In the other mechanism, the C–N bond dissociates to produce a different intermediate complex. The fragmentation of this [p-toluenesulfonamide/carbocation] complex eliminates p-toluenesulfonamide and releases the carbocation (Pathway B). Computations carried out by the Hartree-Fock method suggested that the Pathway B is more favorable. However, a peak for the carbocation is observed only when the carbocation formed is relatively stable. For example, the spectrum of N-phenylethyl-p-toluenesulfonamide is dominated by the peak at m/z 105 for the incipient phenylethyl cation, which rapidly isomerizes to the remarkably stable methylbenzyl cation. The peaks for the carbocations are weak or absent in the spectra of most of N-alkyl-p-toluenesulfonamides because alkyl carbocations, such as the decyl cation, rearrange to more stable secondary cations by 1,2-hydride and alkyl shifts. The energy freed is not dissipated, but gets internalized, causing the carbocation to dissociate either by transferring a proton to the sulfonamide or by releasing smaller alkenes to form smaller carbocations. The loss of the positional integrity in this way was proven by deuterium labeling experiments.

A dramatic “ortho effect” was observed during gas-phase dissociation of ortho-, meta-, and para-methoxybenzoate anions. Upon activation under mass spectrometric collisional activation conditions, anions generated from all three isomers undergo a CO2 loss. Of the m/z 107 ions generated in this way, only the 1-dehydro-2-methoxybenzene anion from the ortho isomer underwent an exclusive formaldehyde loss. A peak for a formaldehyde loss in the spectra of 2,4-, 2,5-, and 2,6-dimethoxybenzoates and the absence of an analogous peak from 3,4- and 3,5-dimethoxy derivatives confirmed that this is a diagnostically useful ortho-isomer-specific phenomenon. Moreover, the spectrum from 2,3-dimethoxybenzoic acid showed peaks for two consecutive formaldehyde losses. The 1-dehydro-2,3,4-trimethoxybenzene anion (m/z 167) generated from 2,3,4-trimethoxybenzoate in this way endures three consecutive eliminations of formaldehyde units. For this, the negative charge, initially located on position 1, circumambulates to position 2, then to position 3, and finally to position 4 to form the final phenyl anion. The proposed stepwise fragmentation pathway, which resembles the well-known E1cB-elimination mechanism, is supported by tandem mass spectrometric observations made with 2-[13C2H3]methoxy-3-[13C]methoxy-4-methoxybenzoic acid, and ab initio calculations. In addition, the spectra of ions such as 1-dehydro-3,4-dimethoxybenzene anion show peaks for consecutive methyl radical losses, a feature that establishes the 1,2-relationship between the two methoxy groups.

Solid HgCl2 is readily detected at ambient conditions by electron capture in a HePI-MS source. The captured electron occupies the empty 6 s orbital of the Hg atom. The resulting radical-anion HgCl2−• can exist as three “flexomers” of different Cl-Hg-Cl angle. The facile in-source formation of HgCl2−• and the adduct [HgCl3]–- is exploited to detect other solid Hg compounds by exposing them to an external chloride source, such as HCl, NaCl, or vapors emanating from solid TiCl3. In situ oxidation of Hg2Cl2 with H2O2 generated signals for HgCl2−• and [HgCl3] –, suggesting that oxidation makes Hg 6 s orbital available for electron capture.

Inorganic nitrates in solid deposits were detected directly by ambient-pressure helium-plasma ionization-mass spectrometry (HePI-MS), without the need for extensive sample preparation. Nitrates were detected even from complex matrices such as meats and fruit juices. Any electrospray-ionization mass spectrometer can be modified to perform ambient-pressure HePI-MS by simply passing helium through the metal capillary intended for liquid-sample delivery. Nitrates on paper strips, glass slides, or cotton swabs (sometimes wetted with a mineral acid) were inserted directly into the ambient-pressure HePI source. The spectra acquired under negative-ion generating conditions showed a peak at m/z 62 for the nitrate ion, along with a lower-intensity peak at m/z 125 for the nitrate adduct of nitric acid. Apparently, it is nitric acid that is initially transferred to the gas phase, forming an ion–molecule complex with hydroxyl anions present in the plasma. The ion-neutral complex then dissociates by eliminating water to produce gaseous NO3– ions. This hypothesis was supported by the observation that certain solid nitrate salts, which were not readily amenable to HePI (notably the alkali nitrates), were immediately detected as m/z 62 and 125 ions upon acidification by a strong acid. Quantitative evaluations showed that the nitrate-signal response versus the deposited mass is linear for over 3 orders of magnitude. With the use of 15N-labeled nitrate (m/z 63), the limit of detection was determined to be as low as 200 fg.

The diagnostic value of the “ortho effect” for unknown identification by mass spectrometry is well known. Here, we report the existence of a novel “meta effect,” which adds to the repertoire of useful mass spectrometric fragmentation mechanisms. For example, the meta-specific elimination pathway described in this report enables unequivocal identification of meta isomers from ortho and para isomers of carboxyanilides. The reaction follows a specific path to eliminate a molecule of meta-benzyne, from the anion produced after the initial decarboxylation of the precursor. Consequently, in the CID spectra of carboxyanilides, a peak for the (R-CO-NH)– anion is observed only for the meta isomers. For example, the peaks observed at m/z 58, 86, 120, 128, and 170 from acetamido-, butamido-, benzamido, heptamido-, and decanamido-benzoates, respectively, were specific only to the spectra of meta isomers.

Chemical-ionization techniques that use metastable species to ionize analytes traditionally use a flat pin or a sharp solid needle onto which the high potential needed to generate the discharge plasma is applied. We report here that direct analysis of samples containing volatile and semivolatile compounds, including saturated and unsaturated aliphatic hydrocarbons, can be achieved on any electrospray-ionization mass spectrometer by passing helium though the sample delivery metal capillary held at a high potential. In the helium plasma ionization source (HPIS) described here, the typical helium flow required (about 20–30 mL/min), was significantly lower than that needed for other helium-ionization sources. By this procedure, positive ions were generated by nominal hydride ion removal from molecules emanating from heated saturated hydrocarbons as large as tetratetracontane (C44H90), at capillary voltages ranging from 2.0 to 4.0 kV. Unsaturated hydrocarbons, on the other hand, underwent facile protonation under much lower capillary voltages (0.9 to 2.0 kV). Although saturated and monounsaturated hydrocarbons bearing the same number of carbon atoms generate ions of the same m/z ratio, a gas-phase deuterium exchange method is described to ascertain the identity of these isomeric ions originating from either protonation or hydride abstraction mechanisms. Moreover, mass spectrometric results obtained by exposing unsaturated hydrocarbons to D2O vapor in an HPIS-MS instrument confirmed that the proton donor for ionization of unsaturated hydrocarbons is protonated water.

A source that couples the desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) techniques together was demonstrated to broaden the range of compounds that can be analyzed in a single mass spectrometric experiment under ambient conditions. A tee union was used to mix the spray reagents into a partially immiscible blend before this mixture was passed through a conventional electrospray (ES) probe capillary. Using this technique, compounds that are ionized more efficiently by the DICE method and those that are ionized better with the DESI procedure could be analyzed simultaneously. For example, hydroquinone, which is not detected when subjected to DESI-MS in the positive-ion generation mode, or the sodium adduct of guaifenesin, which is not detected when examined by DICE-MS, could both be detected in one experiment when the two techniques were combined. The combined technique was able to generate the molecular ion, proton and metal adduct from the same compound. When coupled to a tandem mass spectrometer, the combined source enabled the generation of product ion spectra from the molecular ion and the [M + H]+ or [M + metal]+ ions of the same compound without the need to physically change the source from DICE to DESI. The ability to record CID spectra of both the molecular ion and adduct ions in a single mass spectrometric experiment adds a new dimension to the array of mass spectrometric methods available for structural studies.

Facile generation of series of singly charged radical anions (Sn−·; n=1–15) and cations (Sn+·; n=2–11) by direct laser ionization renders elemental sulfur an excellent material for the low-mass-region calibration of time of flight (TOF) mass spectrometers. Upon irradiation by a 337-nm UV laser, elemental sulfur undergoes facile ionization without the need of an additional laser-absorbing matrix. An intense and evenly spaced set of peaks is obtained in both modes.

An ambient pressure ionization technique for mass spectrometric analysis of substances present on solid surfaces was developed. A nebulized spray containing molecular ions of a solvent such as toluene can be generated by passing the solvent through a stainless steel capillary held at a high voltage. When the stream of charged droplets produced in this way is directed onto a solid surface, the analytes present on the surface are desorbed and ionized by a charge exchange process. This technique was shown to desorb and generate positively charged molecular ions from compounds that are not readily ionized by some other ambient methods, under positive-ion generation mode. For example, intense signals representing radical cations of 1,4-hydroquinone, limonene, thymol, and several other compounds were observed when the analytes were deposited on a metal surface and exposed to a toluene spray nebulized from the metal capillary maintained at a potential of about +5 kV. In contrast, when the same samples were exposed to a spray of water/methanol/formic acid under customary DESI-like (positive-ion mode) conditions, no peaks representing the analytes were observed.

The presence of a peak centered near m/z 2862, observed for the first time for the caged dodecatungstate radical-anion, [W12O41]−·, enables distinguishing WO2 from WO3 by Laser Desorption Ionization mass spectrometry (LDI-MS). In addition to WO2, laser irradiation of dry deposits made from aqueous ammonium paratungstate, and calcium and lead orthotungstate also produce the [W12O41]−·. In contrast, spectra recorded from deposits made from aqueous Na2WO4, sodium metatungstate, and WO3, or non-aqueous calcium and lead orthotungstate, and ammonium paratungstate, failed to show the m/z 2862 peak cluster. These observations support the hypothesis that polycondensation reactions to form [W12O41]−· occur solely in the presence of water. Although dry spots are irradiated for ionization, the solvent used for sample preparation plays an important role on the chemical composition endowed to ions detected. For example, the m/z 2862 peak seen from deposits made from aqueous ammonium paratungstate, and calcium and lead orthotungstate, is absent in the spectra recorded either from pristine deposits or those derived from solutions made with organic solvents such as acetonitrile or ethanol.

The major constituent in the pygidial gland defensive fluid of the carabid beetle Ardistomis schaumii is (R)-(+)-limonene, whereas that of Semiardistomis puncticollis is (S)-(−)-limonene. This was an unanticipated result, since it is not very common to find the opposite enantiomers of the same compound among the secondary metabolites of related species. Moreover, the glandular liquid of A. schaumii contains 1,8-cineole, and that of S. puncticollis has β-pinene, β-phellandrene, sabinene, and p-cymene. Of about 500 carabid species that have been chemically investigated, this is the first report of the presence of such complex mixtures of monoterpenes in their defensive secretions.

Electron ionization (EI) mass spectra are not very helpful for characterizing ortho, meta, and para isomers of underivatized haloanilines since their spectra are virtually identical. In contrast, when the amino group of chloro-, bromo-, or iodoanilines is transformed to an N-formyl, N-acetyl, or N-benzoyl derivative, the spectra of the derivatives reveal a highly dramatic loss of a halogen radical, instead of an HX elimination usually expected from an “ortho effect. ” For example, the spectra of N-formyl, N-acetyl, and N-benzoyl derivatives of ortho isomers of chloro-, bromo-, and iodoanilines show a very prominent peak at m/z 120, 134, and 196, respectively, for the loss of the corresponding halogen atom.

Several volatile compounds, including terpenoids, fatty alcohols, fatty acids and some of their esters, were identified from solvent extracts prepared from anal scent glands of nutria (a.k.a. coypu), a serious rodent pest ravaging wetlands in the USA. The major terpenoid constituents were identified as (E,E)-farnesol and its esters by a comparison of their gas chromatographic retention times, and electron-ionization (EI) and chemical-ionization (CI) mass spectra with those of authentic compounds. EI mass spectra of the four farnesol isomers are very similar, however, the ChemStation (Agilent) and GC–MS Solution (Shimadzu) software algorithms were able to identify the natural compound as the (E,E)-isomer, when a high-quality mass spectral library was compiled from reference samples and used for searching. Similarly, the esters were identified as those of (E,E)-farnesol. In contrast to EI spectra, the CI spectra of the (E,E)- and (E,Z)-isomers are distinctly different from those of the (Z,E)- and (Z,Z)-isomers. The intensities (I) of the peaks for the m/z 137 and 121 ions in the CI spectra offer a way of determining the configuration of the C-2 double bond of farnesols (for 2E isomers I137 > I121, whereas for 2Z isomers I137 < I121). Moreover, the infrared spectrum of the (E,E)-isomer is distinctly different from those of the other three isomers in the 2962–2968 cm−1 and 2918–2922 cm−1 bands, which represent asymmetric CH3 and CH2 stretching vibrations, respectively. Finally, the GC retention indices of farnesol and farnesyl ester isomers determined from authentic samples were used to confirm all identifications.

The presence of considerable quantities of hydroquinones including hydroquinone, 2-methylhydroquinone, 2,3- dimethoxyhydroquinone, 2-methoxy-3-methylhydroquinone, and 2,3-dimethoxy-5-methylhydroquinone renders the defensive secretion of Acladocricus setigerus (Silvestri, 1897) significantly different from that of other quinone-producing millipedes. In addition, two hitherto undescribed compounds, namely, 2,3-dimethoxy-5-methylhydroquinone and 2-methyl-3,4-methylenedioxyphenol, were characterized from the defensive secretion. However, it is uncertain if the latter compound is formed after the release of the secretion. The two major compounds, 2-methyl-1,4-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone, constitute about 75% of the defensive fluid. Furthermore, hydrocarbons, which are typically present in the secretions of most other arthropods that use benzoquinones as repellents, are notably absent in the secretion of A. setigerus.

Tiglic, 2-methylbutyric, and ethacrylic acids are found in the pygidial gland defensive fluid of many carabid beetles. By injecting a deuterium-labeled precursor into the carabid beetle Pterostichus (Hypherpes) californicus, and analyzing the defensive fluid by gas chromatography/mass spectrometry, we were able to demonstrate that tiglic and ethacrylic acids are biosynthesized from isoleucine via 2-methylbutyric acid. Moreover, we observed that the injection of l-isoleucine induces an increased production of tiglic acid in P. californicus. A strong primary kinetic isotope effect was found to operate in the dehydrogenation step of 2-methylbutyric acid to tiglic and ethacrylic acids. Consequently, ethacrylic acid was found to preferentially accumulate the deuterium labeling from [2,3,4,4-2H4]isoleucine during our biosynthetic experiments.

An ambient pressure ionization technique for mass spectrometric analysis of substances present on solid surfaces was developed. A nebulized spray containing molecular ions of a solvent such as toluene can be generated by passing the solvent through a stainless steel capillary held at a high voltage. When the stream of charged droplets produced in this way is directed onto a solid surface, the analytes present on the surface are desorbed and ionized by a charge exchange process. This technique was shown to desorb and generate positively charged molecular ions from compounds that are not readily ionized by some other ambient methods, under positive-ion generation mode. For example, intense signals representing radical cations of 1,4-hydroquinone, limonene, thymol, and several other compounds were observed when the analytes were deposited on a metal surface and exposed to a toluene spray nebulized from the metal capillary maintained at a potential of about +5 kV. In contrast, when the same samples were exposed to a spray of water/methanol/formic acid under customary DESI-like (positive-ion mode) conditions, no peaks representing the analytes were observed.A nebulized spray containing molecular ions of toluene desorbs and generates gaseous molecular radical cations from substances present on solid surfaces at ambient pressure.Download high-res image (119KB)Download full-size image

A simple modification converts an electrospray ion source to an ambient-pressure helium plasma ionization source without the need of additional expensive hardware. Peaks for active ingredients were observed in the spectra recorded from intact pharmaceutical tablets placed in this source. A flow of heated nitrogen was used to thermally desorb analytes to gas phase. The desorption temperatures were sometimes as low as 50 °C. For example, negative-ion spectra recorded from an aspirin tablet showed peaks at m/z 137 (salicylate anion) and 179 (acetylsalicylate anion) which were absent in the background spectra. The overall ion intensity increased as the desorption gas temperature was elevated. Within the same acquisition experiment, both positive- and negative-ion signals for acetaminophen were recorded from volatiles emanating from Tylenol tablets by switching the polarity of the capillary back and forth. Moreover, different preparations of acetaminophen tablets could be distinguished by their ion-intensity thermograms.

Electron ionization (EI) mass spectra are not very helpful for characterizing ortho, meta, and para isomers of underivatized haloanilines since their spectra are virtually identical. In contrast, when the amino group of chloro-, bromo-, or iodoanilines is transformed to an N-formyl, N-acetyl, or N-benzoyl derivative, the spectra of the derivatives reveal a highly dramatic loss of a halogen radical, instead of an HX elimination usually expected from an “ortho effect.” For example, the spectra of N-formyl, N-acetyl, and N-benzoyl derivatives of ortho isomers of chloro-, bromo-, and iodoanilines show a very prominent peak at m/z 120, 134, and 196, respectively, for the loss of the corresponding halogen atom.