Qualitative analysis of some carboxylic acids by ion-exclusion chromatography with atmospheric pressure chemical ionization mass spectrometric detection

A simple, selective and sensitive method for the determination of carboxylic acids has been developed. A mixture of formic, acetic, propionic, valeric, isovaleric, isobutyric, and isocaproic acids has been separated on a polymethacrylate-based weak acidic cation-exchange resin (TSK gel OA pak-A) based on an ion-exclusion chromatographic mechanism with detection using UV-photodiode array, conductivity and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). A mobile phase consisting of 0.85 mM benzoic acid in 10% aqueous methanol (pH 3.89) was used to separate the above carboxylic acids in about 40 min. For LC-MS, the APCI interface was used in the negative ionization mode. Linear plots of peak area versus concentration were obtained over the range 1-30 mM (r2=0.9982) and 1-30 mM (r2=0.9958) for conductimetric and MS detection, respectively. The detection limits of the target carboxylic acids calculated at S/N=3 ranged from 0.078 to 2.3 microM for conductimetric and photometric detection and from 0.66 to 3.82 microM for ion-exclusion chromatography-APCI-MS. The reproducibility of retention times was 0.12-0.16% relative standard deviation for ion-exclusion chromatography and 1.21-2.5% for ion-exclusion chromatography-APCI-MS. The method was applied to the determination of carboxylic acids in red wine, white wine, apple vinegar, and Japanese rice wine.     

Reactivity of acetonyl anion with nitroaromatics: an atmospheric pressure chemical ionization study

Nucleophilic attack by the acetonyl anion (CH₃COCH) on highly electron‐deficient nitroaromatics such as trinitrotoluene (TNT) results in the carbon‐bonded anionic σ‐complexes (Meisenheimer complexes). The complexes are generated at atmospheric pressure with relatively low internal energy and do not dissociate under atmospheric pressure chemical ionization conditions, but can be dissociated upon collisional activation to produce characteristic nitrobenzyl‐substituted acetonyl anions via elimination of HNO₂. Further fragmentation produces deprotonated nitrotoluene anions through the loss of CH₂CO, while loss of CH₂COCH₃ and CH₃COCH₃ yields nitroaromatic molecular anions and their H‐loss counterparts. Hydrogen/deuterium (H/D) scrambling is observed in the fragmentation products of the [TNT · CD₃COCD₂]^? complex to extents which vary for different fragmentation pathways. Selective Meisenheimer complex formation, together with its distinctive fragmentation pattern, supplies a highly discriminatory method for detection of TNT and related compounds. Copyright © 2005 John Wiley & Sons, Ltd.     

Unusual atmospheric pressure chemical ionization conditions for detection of organic peroxides

Organic peroxides such as the cumene hydroperoxide I (M(r) = 152 u), the di-tert-butyl peroxide II (M(r) = 146 u) and the tert-butyl peroxybenzoate III (M(r) = 194 u) were analyzed by atmospheric pressure chemical ionization mass spectrometry using a water-methanol mixture as solvent with a low flow-rate of mobile phase and unusual conditions of the source temperature (< or =50 degrees C) and probe temperature (70-200 degrees C). The mass spectra of these compounds show the formation of (i) an [M + H](+) ion (m/z 153) for the hydroperoxide I, (ii) a stable adduct [M + CH(3)OH(2)](+) ion (m/z 179) for the dialkyl peroxide II and (iii) several protonated adduct species such as protonated molecules (m/z 195) and different protonated adduct ions (m/z 227, 389 and 421) for the peroxyester III. Tandem mass spectrometric experiments, exact mass measurements and theoretical calculations were performed for characterize these gas-phase ionic species. Using the double-well energy potential model illustrating a gas-phase bimolecular reaction, three important factors are taken into account to propose a qualitative interpretation of peroxide behavior toward the CH(3)OH(2) (+), i.e. thermochemical parameters (DeltaHdegrees(reaction)) and two kinetic factors such as the capture constant of the initial stable ion-dipole and the magnitude of the rate constant of proton transfer reaction into the loose proton bond cluster.     

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: A comparison of atmospheric pressure chemical ionization and electrospray ionization

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Comparison of the repeatability of quantitative data measured in high-performance liquid chromatography with UV and atmospheric pressure chemical ionization mass spectrometric detection

A detailed comparison of the repeatability of the retention times, the peak efficiencies and the peak areas of a dozen probe compounds achieved in HPLC, using either HPLC-UV or HPLC-MS for detection purpose, is reported. Three groups of conventional analytes, each one separated under a different set of experimental conditions, were selected for this study. Most of the compounds are basic, the other ones being neutral. The repeatabilities of the retention times do not exhibit any influence of the mode of detection. However, the repeatabilities of the peak areas and the column efficiencies are generally (although not always) better in HPLC-UV than that in HPLC-MS. On average, the precision for the UV peak area detection was 2.5% versus 6.8% for MS detection. Experimental results show that the response factor of the UV detector is more constant than that of the MS detector, probably because the HPLC flow-rate was sufficiently stable. The results obtained in the different tests are discussed.     

Gas phase anion reactions at atmospheric pressure : Atmospheric pressure ionization mass spectrometric studies related to the electron-capture detector

Negative ion atmospheric pressure ionization mass spectrometry has been used to investigate the gas phase atmospheric pressure anion chemistry of N₂O₂H⁻ and . N₂O₂H⁻ has been shown to be a stronger base than . Specific types of reaction (e.g. proton abstraction, and dehydration) have been identified for each of these anions. Although the analytical significance of these reactions has not yet been demonstrated, certain compounds such as alcohols which do not readily attach electrons directly can easily be detected by observing a specific anion reaction product. The technique appears to provide an additional dimension to established gas chromatographic—mass spectrometric analyses.     

Separation of triacylglycerols in a complex lipidic matrix by using comprehensive two-dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection

The present investigation describes the employment of a comprehensive 2-D HPLC system, based on the combination of a silver ion and an RP column, for the characterization of the triacylglycerol (TAG) fraction of a very complex lipidic sample: donkey milk fat. The TAGs were grouped on the resulting bidimensional contour plot according to their double bond numbers (aligned along vertical bands) and according to their partition numbers (aligned along horizontal bands). Peak assignment was supported by using atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection. The combination of the enhanced resolving power of comprehensive multidimensional LC, the formation of ordered 2-D patterns, and APCI-MS detection proved to be an effective tool for the characterization of the complex matrix, enabling the separation and identification of nearly 60 TAGs.     

Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.     

Rapid detection of drugs in biofluids using atmospheric pressure chemi/chemical ionization mass spectrometry

We have demonstrated that, with simple pH adjustment, volatile drugs such as methamphetamine, amphetamine, 3,4‐methylenedioxymethamphetamine (MDMA), ketamine, and valproic acid could be analyzed rapidly from raw biofluid samples (e.g. urine and serum) without dilution, or extraction, using atmospheric pressure ionization. The ion source was a variant type of atmospheric pressure chemical ionization (APCI) that used a dielectric barrier discharge (DBD) to generate the metastable helium gas and reagent ions. The sample solution was loaded in a disposable glass pipette, and the volatile compounds were purged by nitrogen gas to be reacted with the metastable helium gas. The electrodes of the DBD were arranged in such a way that the generated glow discharge was confined within the discharge tube and was not exposed to the analytes. A needle held at 100–500 V was placed between the ion‐sampling orifice and the discharge tube to guide the analyte ions into the mass spectrometer. After pH adjustment of the biofluid sample, the amphiphilic drugs were in the form of a water‐insoluble oil, which could be concentrated on the liquid surface. By gentle heating of the sample to increase the evaporation rate, rapid and sensitive detection of these drugs in raw urine and serum samples could be achieved in less than 2 min for each sample. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of sulfonamides by packed column supercritical fluid chromatography with atmospheric pressure chemical ionisation mass spectrometric detection

Sulfonamide antibiotics are widely used to prevent bacterial infections in livestock, and residues are commonly found in milk and meat. Packed column supercritical fluid chromatography (pSFC) with detection using ultra violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the detection and quantification of six major sulfonamides. The APCI mass spectra for all the sulfonamides consisted of protonated molecules at low cone voltages. Increasing the cone voltage led to informative fragmentation patterns, which provided structural information for identification purposes. The pSFC-APCI-MS technique was shown to be linear (r2 > or = 0.999) over the concentration range 0.1-50 micrograms ml-1 using total ion current. The precision and the accuracy of the system and validation of sample preparation are acceptable, with RSD < 2% and relative error 8%. Selected ion monitoring gave detection limits as follows: sulfadiazine 41, sulfamethoxazole 45, sulfamerazine 47, sulfamethizole 59, sulfamethazine 181 and sulfadimethoxine 96 micrograms l-1, which are lower than the amounts permitted in milk products. The APCI pSFC-MS system was shown to have a high degree of reproducibility. The technique was then applied to determine the above sulfonamides in milk. The results obtained show that there are no matrix effects from the milk and that the detection limits remained as stated for the standard solutions.     

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.     

On-line characterization of gaseous and particulate organic analytes using atmospheric pressure chemical ionization mass spectrometry

A modified atmospheric pressure chemical ionization ion source is applied for direct analysis of volatile or low volatile organic compounds in air. The method is based on the direct introduction of the analytes in the gas phase and/or particle phase into the ion source of a commercial ion-trap mass spectrometer. Two methods are employed for the production of primary ions at atmospheric pressure, photoionization and corona discharge. It is shown that in the presence of a dopant, photoionization can be a highly efficient ionization method also for real-time analysis with detection limits for selected analytes in the lower ppt-range. Using corona discharge for the production of primary ions, which is instrumentally easier since no additional chemicals have to be added to the sample flow, we demonstrate the analytical potential of on-line atmospheric pressure chemical ionization mass spectrometry for reaction monitoring experiments. To do so, an atmospherically relevant gas phase reaction is carried out in a 500 l reaction chamber and gaseous and particulate compounds are monitored in the positive and negative ion mode of the mass spectrometer.     

Formation of solvated ions in the atmospheric interface of an electrospray ionization triple-quadrupole mass spectrometer

A simple method capable of generating and investigating various solvent clusters and solvated ions was developed. The technique opens a door to studying these complexes on commercially available instruments. Formation of the desired solvated ion in the gas phase was achieved by introducing the appropriate volatile solvent vapour into the curtain gas stream. Capabilities of the technique are illustrated by generating alkali, alkaline earth and transition metal cations solvated by various volatile compounds such as water, methanol and acetonitrile. Depending on the ligands and on the experimental conditions, clusters of 2-100 molecules may be observed. Isotope labelling suggests that these are formed by a re-solvation process in the curtain gas region.     

Unexpected observation of ion suppression in a liquid chromatography/atmospheric pressure chemical ionization mass spectrometric bioanalytical method

Ion suppression is a well-known phenomenon in electrospray ionization (ESI) mass spectrometry. These suppression effects have been shown to adversely affect the accuracy and precision of quantitative bioanalytical methods using ion spray. Such suppression effects have not been as well defined in atmospheric pressure chemical ionization (APCI) and there is some debate whether these effects actually occur in the ionization process using APCI. Here an example is described where clear ion suppression was observed during studies on a model compound and three metabolites using APCI liquid chromatography/tandem mass spectrometry (LC/MS/MS).     

Chloride-enhanced atmospheric pressure chemical ionization mass spectrometry of polychlorinated n-alkanes

The use of high-performance liquid chromatography combined with chloride-enhanced atmospheric pressure chemical ionization for the determination of polychlorinated n-alkanes (PCAs, also called chlorinated paraffins or CPs) is described as an alternative to gas chromatographic methods. Atmospheric pressure chemical ionization in the negative ion mode formed exclusively [M+Cl](-) adduct ions and suppressed fragmentation when a chlorinated solvent was added. Limits of detection were 1-2 ng/microL for technical PCA mixtures. Response factors for single short-chain PCA homologues with different degrees of chlorination varied by not more than a factor of 6.5. The developed method was applied for the determination of the composition of technical PCA mixtures as well as for the analysis of PCAs in household commodities. Medium-chain PCAs were found in paint samples at concentrations of 8.2-11.5% (w/w), compared with 7.4-11.5% obtained by gas chromatography combined with electron ionization tandem mass spectrometry.     

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.     

Reaction of analyte ions with neutral chemical ionization gas

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ion-molecule reactions of neutral methane with analyte ions under normal methane chemical ionization conditions are discussed. Reactant ions can be generated by direct electron ionization (EI) fragmentation, chemical ionization (CI) fragmentation, or collision-induced dissociation (CID). Examples in which products of such reactions appear in mass spectra in both conventional CI sources in “beam” instruments and low pressure CI in a quadrupole ion trap are presented. Also shown is an example in which MS/MS product ions react with neutral methane used for CI in an ion trap. It is shown that it is relatively straightforward to recognize such reactions in a quadrupole ion trap and in certain cases to minimize or preclude them. Effects of various operating parameters have been investigated and are discussed.