Use of electrospray ionization mass spectrometry for the investigation of radical cation chain reactions in solution: detection of transient radical cations

Electrospray ionization mass spectrometry (ESI-MS) coupled to a microreactor is an excellent tool for the investigation of reactions in solution. Here, we report the first results of our investigations into preparatively interesting electron-transfer-initiated chain reactions in solution which proceed via radical cations as reactive intermediates. The tris(p-bromophenyl)aminium hexachloroantimonate (1)-mediated [2+2] cycloaddition of trans-anethole (2) to give 1,2-bis(4-methoxyphenyl)-3,4-dimethylcyclobutane (3) was investigated. The reaction proceeds as a radical cation chain reaction via transient intermediates 2 ⁺ and 3 ⁺ that could be detected and characterized unambiguously directly in the reacting solution by ESI-MS/MS. The identity of the intermediates was confirmed by comparison with authentic MS/MS spectra of 2 ⁺ and 3 ⁺ obtained by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). In addition, substrate and product can be monitored easily in the reacting solution by APCI-MS.Part of this work was presented at the 36th Conference of the German Society for Mass Spectrometry (DGMS).     

Accelerated reactions in field desorption mass spectrometry

Field desorption mass spectrometry under ambient conditions is used to study solution‐phase organic reactions in micro‐volumes. Reagent solution is transferred onto the microdendrites of the field emitter, and reaction products are examined online by mass spectrometry. Three reactions, hydrazone formation by phenyl hydrazine and indoline‐2,3‐dione, the Katritzky reaction between a pyrylium salt and anisidine, and the Hantzsch synthesis of 1,4‐dihydropyridine, were investigated, and reaction acceleration was observed to different extents. The increase in rate relative to the corresponding bulk reactions is attributed to solvent evaporation (simple concentration effect) and to the increase of surface‐to‐volume ratio (enhanced interfacial reactions). A distinguishing feature of this method of reaction acceleration, relative to that based on nano electrospray ionization, is the observation of radical cations and the formation of radical cation products. The study also breaks new ground in using field emitters at atmospheric pressure.     

Laser-based ion mobility spectrometer for the direct analysis of aromatic compounds in liquids

A laser-based ionization source for the direct analysis of liquid samples in ion mobility (IM) spectrometry is presented and characterized. Ionization of aromatic substances in liquids is achieved, analogous to atmospheric pressure laser ionization (APLI) in mass spectrometry, by vaporizing the liquid and subsequently ionizing the aromatic substances by resonance-enhanced multiphoton ionization (REMPI). The effects of parameters, such as composition and flow rate of the solvent as well as laser wavelength and pulse energy, are systematically investigated. The characterization of the IM spectrometer is carried out by means of selected substances from diverse fields of applications, e.g., polycyclic aromatic hydrocarbons (PAH), pesticides, wood preservatives and drug compounds. Limits of detection (LOD) down to 10 fmol and linear ranges up to three orders of magnitude are established. In addition to direct laser ionization, indirect laser ionization via dopants (toluene) for substances with low ionization efficiencies is investigated. Ionization occurs as a result of proton transfer from toluene radical cations to substances of sufficiently high proton affinities. As a result of indirect laser ionization, LOD could be decreased by up to two orders of magnitude. Ionization products are investigated by means of a combination of IM and mass spectrometer. Depending on the substance investigated primary ions (radical cations) and secondary ions (protonated molecules) resulting from ion molecule reactions are formed.     

Characterization of amiodarone metabolites and impurities using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

Using the high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS) technique, together with established trends from the literature, the structures of metabolites and impurities of amiodarone, an anti-arrhythmic drug, have been assigned. By comparing analyses of products of incubation with rat liver microsomes with controls in which glucose 6-phosphate dehydrogenase was omitted, metabolites could be distinguished from impurities. Structures for the two proposed metabolites and four impurities are proposed.     

Liquid chromatography–electrochemistry–mass spectrometry of polycyclic aromatic hydrocarbons

An efficient method for fast elucidation of the electrochemical reactions of polycyclic aromatic hydrocarbons (PAH) has been set up by applying post-column electrochemistry in liquid chromatography–mass spectrometry (LC–MS). With this set-up strong improvement of sensitivity in the LC–MS analysis of PAH is observed. Due to their low redox potentials, the non-polar PAH are converted into the respective radical cations, which may further react with constituents of the mobile phase and in additional electrochemical oxidation steps. Among other products, mono-, di-, and trioxygenated species are observed in aqueous solutions, alkoxylated compounds in alcohols, and solvent adducts in the presence of acetonitrile. While more different products are observed by using atmospheric pressure chemical ionization in the positive-ion mode (APCI(+)), the deprotonation of hydroxylated species results in very clear spectra in the negative-ion mode (APCI(–)). Deuterated PAH and deuterated solvents were used to gain additional information on the formation of the reaction products.     

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products

Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.     

Non-volatile products of triolein produced at frying temperatures characterized using liquid chromatography with online mass spectrometric detection.

Oxidation products from triolein under model heated frying conditions have been analyzed using liquid chromatography with an evaporative light scattering detector and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Triolein was heated at 190 degrees C with 2% water added each hour, to simulate the moisture of a frozen product, until polar components reached approximately 30%. The samples were separated using reversed-phase high-performance liquid chromatography with APCI-MS detection. Triolein oxidation products included hydroperoxides, epoxides and a ketone. Other products were formed by shortening of an acyl chain on the intact triolein. Normal and oxygen-containing products formed by the dimerization of triolein were also observed. Other products included chain addition products formed by addition of acyl chain subunits to intact triolein to form higher molecular weight products.     

A new approach for the study of gas-phase ion-ion reactions using electrospray ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)₃) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]ⁿ⁺ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.     

Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection.

In this paper the application of on-line HPLC-UV-APCI (atmospheric pressure chemical ionization) mass spectrometry (MS) coupling for the separation and determination of different carotenoids as well as cis/trans isomers of beta-carotene is reported. All HPLC separations were carried out under RP conditions on self-synthesized polymeric C30 phases. The analysis of a carotenoid mixture containing astaxanthin, canthaxanthin, zeaxanthin, echinenone and beta-carotene by HPLC-APCI-MS was achieved by scanning the mass range from m/z 200 to 700. For the characterization of a sample containing cis/trans isomers of beta-carotene as well as their oxidation products, a photodiode-array UV-visible absorbance detector was used in addition between the column and the mass spectrometer for structural elucidation of the geometrical isomers. The detection limit for beta-carotene in positive-ion APCI-MS was determined to be 1 pmol. In addition, an extract of non-polar substances in vegetable juice has been analyzed by HPLC-APCI-MS. The included carotenoids could be identified by their masses and their retention times.     

Lateral extension of π conjugation along the bay regions of bisanthene through a Diels-Alder cycloaddition reaction

Diels-Alder cycloaddition reactions at the bay regions of bisanthene (1) with dienophiles such as 1,4-naphthoquinone have been investigated. The products were submitted to nucleophilic addition followed by reductive aromatization reactions to afford the laterally extended bisanthene derivatives 2 and 3. Attempted synthesis of a larger expanded bisanthene 4 revealed an unexpected hydrogenation reaction at the last reductive aromatization step. Unusual Michael addition was observed on quinone 14, which was obtained by Diels-Alder reaction between 1 and 1,4-anthraquinone. Compounds 1-3 exhibited near-infrared (NIR) absorption and emission with high-to-moderate fluorescent quantum yields. Their structures and absorption spectra were studied by density function theory and non-planar twisted structures were calculated for 2 and 3. All compounds showed amphoteric redox behavior with multiple oxidation/reduction waves. Oxidative titration with SbCl(5) gave stable radical cations, and the process was followed by UV/Vis/NIR spectroscopic measurements. Their photostability was measured and correlated to their different geometries and electronic structures.     

Supercritical fluid extraction and quantification of aflatoxins in Zizyphi Fructus by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

An integrated method combining supercritical fluid extraction (SFE) with liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS) was developed and successfully applied to quantify aflatoxins (AFs) in Zizyphi Fructus (fruits of Zizyphus jujube), a traditional Chinese medicine. To minimize the potential interferences caused by the complex matrix in Zizyphi Fructus, a SFE pretreatment was performed. In addition, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) spectra were also compared. The results showed that the calibration curves of AFB(1), AFB(2), AFG(1), and AFG(2) were all linear over the range of concentration from 1 to 50 ng/g, the squared correlation coefficients (r(2)) were over 0.995, and the detection limits of the method were between 0.17 and 0.32 ng/g. It showed high recovery and good precision in quantitating AFs in Zizyphi Fructus without further clean-up. Further, fragmentation pathways of protonated AFs in APCI-MS/MS were clearly proposed which could predict the existence of AFB or AFG series. To test the empirical validity of the proposed methodology in this paper, eight random samples of Zizyphi Fructus collected from supermarkets and traditional Chinese medicine stores in different geographical areas of Taiwan were analyzed. The results indicated that low levels of AFs were detected in only one of them.     

Analysis of derivatized and underivatized theanine enantiomers by high-performance liquid chromatography/atmospheric pressure ionization-mass spectrometry

Theanine, a naturally occurring non-proteinic amino acid found in tea leaves, has demonstrated wide-ranging physiological activity, from lowering blood pressure to enhancing the anti-tumor activity of chemotherapeutic drugs. The chiral nature of theanine suggests that enantiospecificity plays a significant role in its various pharmacological functions. Using the Chirobiotic T (teicoplanin) chiral stationary phase, native and derivatized theanine enantiomers were separated and detected via high-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization mass spectrometry (API-MS). With the use of flow rates compatible with each ionization source, native theanine standards achieved excellent sensitivity and detection limits (10 ng/mL) for both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Optimum sensitivity and detection limits for derivatized theanine standards were achieved using ESI-MS. The enantiomeric composition of six commercially available L-theanine samples was evaluated using the high-flow APCI-MS method and confirmed with photodiode array detection. Five of the six products contained significant amounts of D-theanine. Only one product, SunTheanine, appeared to contain only the L-theanine enantiomer.     

Atmospheric pressure ionization mass spectrometry of purine and pyrimidine markers of inherited metabolic disorders

Purines and pyrimidines are of interest owing to their significance in processes in living organisms. Mass spectrometry is a promising analytical tool utilized in their analysis. Two atmospheric pressure ionization (API) methods (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) in both negative and positive modes applied to selected purine and pyrimidine metabolites (markers of inherited metabolic disorders) were studied. APCI is less sensitive to alkali metal cations present in a sample and offers higher response than ESI for studied compounds. Both of the techniques afford quasi-molecular ions, but fragmentation also occurs to a certain extent. However, the application of collision-induced dissociation of quasi-molecular ions is essential to confirm a certain metabolite in a sample. Fragmentation of both positive and negative ions was evaluated using multi-stage mass spectrometric experiments. Typical neutral losses correspond to molecules NH(3), H(2)O, HCN, CO, H(2)NCN, HNCO and CO(2). The ion [NCO](-) arises in the negative mode. The cleavage of the glycosidic C-N bond is characteristic for relevant metabolites. Other neutral losses (CH(2)O, C(2)H(4)O(2) and C(3)H(6)O(3)) originate from fragmentation of the glycosidic part of the molecules. In addition to fragmentation, the formation of adducts of some ions with applied solvents (H(2)O, CH(3)OH) was observed. The composition of the solution infused into the ion source affects the appearance of the mass spectra. Tandem mass spectra allow one to distinguish compounds with the same molecular mass (uridine-pseudouridine and adenosine-2'-deoxyguanosine). Flow injection analysis APCI-MS/MS was tested on model samples of human urines corresponding to adenosine deaminase deficiency and xanthine oxidase deficiency. In both cases, the results showed potential diagnostic usefulness.     

High-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry determination of cholesterol uptake by Caco-2 cells

A simple, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method (LC/APCI-MS/MS) was developed and applied to quantitative determination of uptake of cholesterol by Caco-2 human intestine cells. Caco-2 cells were cultured in medium containing cholesterol-3,4-13C2 and phytosterols from nutritional supplements after in vitro digestion. Cellular cholesterol (cholesterol-3,4-13C2) and endogenous cholesterol were extracted using methanol/chloroform (1:2, v/v) and directly analyzed using LC/APCI-MS/MS with selected reaction monitoring (SRM), using cholesterol-2,2,3,4,4,6-d6 as an internal standard. Detection and quantification limits were 2.2 and 7.2 pmol, respectively. This method provides an effective tool for rapid determination of cholesterol uptake by cells with increased selectivity and sensitivity in comparison to previously reported LC/APCI-MS analysis using selected ion monitoring (SIM).     

Mass spectral fragmentation reactions of a therapeutic 4-azasteroid and related compounds

Mass spectra were acquired for a therapeutic 4-azasteroid (dutasteride), and some related compounds, using various ionization conditions (EI, CI, APCI and ESI) in both positive and negative ion modes. The ionization and fragmentation behavior of the compound dutasteride, its precursors and several analogs is reported. Positive atmospheric pressure chemical ionization (APCI+) and positive electrospray ionization (ESI+) produced distinctive collision-induced dissociation (CID) spectra for the respective [MH]+ ions of dutasteride. The spectral differences are attributed to ion populations having either different structures or different internal energy distributions (as a consequence of the method of ionization). Irrespective of their origin, the protonated molecules undergo interesting fragmentation reactions when collisionally activated. The identity of the major fragmentation products was confirmed by accurate mass measurement. The negative APCI mass spectrum of dutasteride displays extensive dehydrohalogenation, apparently due to the thermal component of the APCI process. Some of the resulting radical anions display remarkable stability toward collisional decomposition. Details of the fragmentation behavior for the negative ion species and their relationship to the positive ion results are discussed.     

Laser flash photolysis kinetic studies of enol ether radical cations. Rate constants for heterolysis of alpha-methoxy-beta-phosphatoxyalkyl radicals and for cyclizations of enol ether radical cations

Two series of enol ether radical cations were studied by laser flash photolysis methods. The radical cations were produced by heterolyses of the phosphate groups from the corresponding alpha-methoxy-beta-diethylphosphatoxy or beta-diphenylphosphatoxy radicals that were produced by 355 nm photolysis of N-hydroxypryidine-2-thione (PTOC) ester radical precursors. Syntheses of the radical precursors are described. Cyclizations of enol ether radical cations 1 gave distonic radical cations containing the diphenylalkyl radical, whereas cyclizations of enol ether radical cations 2 gave distonic radical cation products containing a diphenylcyclopropylcarbinyl radical moiety that rapidly ring-opened to a diphenylalkyl radical product. For 5-exo cyclizations, the heterolysis reactions were rate limiting, whereas for 6-exo and 7-exo cyclizations, the heterolyses were fast and the cyclizations were rate limiting. Rate constants were measured in acetonitrile and in acetonitrile solutions containing 2,2,2-trifluoroethanol, and several Arrhenius functions were determined. The heterolysis reactions showed a strong solvent polarity effect, whereas the cyclization reactions that gave distonic radical cation products did not. Recombination reactions or deprotonations of the radical cation within the first-formed ion pair compete with diffusive escape of the ions, and the yields of distonic radical cation products were a function of solvent polarity and increased in more polar solvent mixtures. The 5-exo cyclizations were fast enough to compete efficiently with other reactions within the ion pair (k approximately 2 x 10(9) s(-1) at 20 degrees C). The 6-exo cyclization reactions of the enol ether radical cations are 100 times faster (radical cations 1) and 10 000 times faster (radical cations 2) than cyclizations of the corresponding radicals (k approximately 4 x 10(7) s(-1) at 20 degrees C). Second-order rate constants were determined for reactions of one enol ether radical cation with water and with methanol; the rate constants at ambient temperature are 1.1 x 10(6) and 1.4 x 10(6) M(-1) s(-1), respectively.     

Application of chiral cationic catalysts to several classical syntheses of racemic natural products transforms them into highly enantioselective pathways

This paper describes the application of chiral oxazaborolidinium cations of type 2 to various enantioselective Diels-Alder reactions that have served as early key steps for the syntheses of complex natural products. In the original syntheses these Diels-Alder reactions produced racemic adducts and led to racemic target molecules unless a separation of enantiomers by classical resolution was employed. By use of chiral catalysts of type 2, chiral products were obtained directly from Diels-Alder reactions of achiral components in excellent yield and enantioselectivity and with the mechanistically predicted absolute configuration. As a result, a number of classical syntheses could be converted to enantioselective versions, including (1) cortisone/cortisol (Merck/Sarett), (2) dendrobine (Kende), (3) vitamin B(12) (Eschenmoser), (4) myrocin C (Chu-Moyer/Danishefsky), (5) coriolin and hirsutene (Mehta), (6) dendrobatid 251F (Aube), (7) silphinene (Ito), and (8) nicandrenone core (Stoltz/Corey).     

Generation of ion-bound solvent clusters as reactant ions in dopant-assisted APPI and APLI

We provide experimental and theoretical evidence that the primary ionization process in the dopant-assisted varieties of the atmospheric pressure ionization methods atmospheric pressure photoionization and atmospheric pressure laser ionization in typical liquid chromatography–mass spectrometry settings is—as suggested in the literature—dopant radical cation formation. However, instead of direct dopant radical cation–analyte interaction—the broadly accepted subsequent step in the reaction cascade leading to protonated analyte molecules—rapid thermal equilibration with ion source background water or liquid chromatography solvents through dopant ion–molecule cluster formation occurs. Fast intracluster chemistry then leads to almost instantaneous proton-bound water/solvent cluster generation. These clusters interact either directly with analytes by ligand switching or association reactions, respectively, or further downstream in the intermediate-pressure regions in the ion transfer stages of the mass spectrometer via electrical-field-driven collisional decomposition reactions finally leading to the predominantly observed bare protonated analyte molecules [M?+?H]⁺.     

液相色谱-大气压化学电离质谱法分析人参中的人参皂甙

研究了用反相高效液相色谱-大气压化学电离质谱(HPLC/APCI-MS)分析人参皂甙的方法。液相色谱采用乙腈-水流动相进行梯度洗脱,质谱采用正负离子同时扫描并结合二级质谱进行定性,用选择反应离子模式(SRM)测定检测限。实验发现虽然人参皂甙是热不稳定物质,但在大气压化学电离质谱的高温汽化过程中仍能检测到很强的负离子分子离子峰,而且随着汽化温度的升高,人参皂甙的负离子分子离子峰的强度增加。该方法对人参皂甙Rb1和Rg1的检测限分别为1.2×10-13 g和3.0×10-14 g,并检测出白参中包括丙二酰人参皂甙在内的29种人参皂甙。该法灵敏度高,重复性好,结果准确,能有效地对药材提取物中的多种人参皂甙进行检测和结构分析。     

Quantitative aspects of and ionization mechanisms in positive-ion atmospheric pressure chemical ionization mass spectrometry

The behavior in atmospheric pressure chemical ionization of selected model polycyclic aromatic compounds, pyrene, dibenzothiophene, carbazole, and fluorenone, was studied in the solvents acetonitrile, methanol, and toluene. Relative ionization efficiency and sensitivity were highest in toluene and lowest in methanol, a mixture of molecular ions and protonated molecules was observed in most instances, and interferences between analytes were detected at higher concentrations. Such interferences were assumed to be caused by a competition among analyte molecules for a limited number of reagent ions in the plasma. The presence of both molecular ions and protonated analyte molecules can be attributed to charge-transfer from solvent radical cations and proton transfer from protonated solvent molecules, respectively. The order of ionization efficiency could be explained by incorporating the effect of solvation in the ionization reactions. Thermodynamic data, both experimental and calculated theoretically, are presented to support the proposed ionization mechanisms. The analytical implications of the results are that using acetonitrile (compared with methanol) as solvent will provide better sensitivity with fewer interferences (at low concentrations), except for analytes having high gas-phase basicities.