Structural characterization of phosphatidylcholines by atmospheric pressure photoionization mass spectrometry

The potential of atmospheric pressure photoionization was investigated for the structural analysis of phosphatidylcholine lipids (PCs). [M+H]+ ions of high abundance were obtained, along with several fragment ions. Three of these dissociation products corresponded to quite unusual fragmentation pathways but allowed the determination of both the nature and the position on the glycerol backbone (sn-1 or sn-2) of the fatty acyl chains. The loss of a methyl group from the choline head was also observed. These results suggest a complex ionization mechanism in APPI. However, this method proved to be very powerful for the rapid structural analysis of PC species without using MS/MS experiments.     

Microemulsion electrokinetic chromatography hyphenated to atmospheric pressure photoionization mass spectrometry

CZE is an appropriate technique for separating charged species, but lacks selectivity for neutral compounds. Alternative approaches such as microemulsion electrokinetic chromatography (MEEKC) have been developed to broaden its range of applications. Hyphenation of MEEKC with MS is an attractive perspective since it can enhance sensitivity and selectivity. The on-line coupling of MEEKC with MS, however, is not straightforward due to the low compatibility of non-volatile surfactant additives (e.g. SDS) and the commonly used API source, namely ESI. In order to hyphenate MEEKC with MS detection, the atmospheric pressure photoionization (APPI) source was investigated. Possibilities offered by the coupling of MEEKC with APPI-MS were highlighted for the complex separation of ionized and neutral compounds in both the positive and negative modes. MEEKC-APPI-MS performance, in terms of selectivity, efficiency and sensitivity was compared to CZE-ESI-MS and MEEKC-ESI-MS for the screening of doping substances (beta-blockers, central stimulants, diuretics, etc). Relevant selectivity and detectability, particularly for neutral, structurally related and isobaric compounds was demonstrated with the MEEKC-APPI-MS approach opening new avenues for CE-MS, in addition to the well-established CZE-ESI-MS technique.     

Analysis of benzylisoquinoline-type alkaloids by electrospray tandem mass spectrometry and atmospheric pressure photoionization

Benzylisoquinoline alkaloids found in the Papaveraceae family play a major role in pharmaceutical biology. This is the first systematic study dealing with electrospray tandem mass spectrometry (ESI-MS/MS) of all benzylisoquinolines found as biogenetic precursors of morphinan alkaloids. Tandem mass spectral data are presented for norlaudanosoline, laudanosoline, 4'-O-methyl-norlaudanosoline, 6-O-methyl-norlaudanosoline, norcoclaurine, coclaurine, N-methylcoclaurine, N-methyl-3'-hydroxycoclaurine, N-methyl-3'-O-methylcoclaurine, norreticuline and reticuline. This study compares results obtained using an ion trap mass spectrometer with those obtained using a triple quadrupole one. The results highlight the differences of the tandem-in-time versus the tandem-in-space principle, often hampering the development of ESI-MS/MS libraries. Additionally, the use of the atmospheric pressure photoionisation technique for the analysis of such substances is discussed.     

Determination of nivalenol and deoxynivalenol by liquid chromatography/atmospheric pressure photoionization mass spectrometry

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high‐performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2–100 ng · mL^?1 was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng · g^?1 and 0.39 ng · g^?1, respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery

The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.     

Dopant-assisted atmospheric pressure photoionization mass spectrometry of polyisobutylene derivatives initiated by mono- and bifunctional initiators

Nine polyisobutylene (PIB) derivatives with different end groups (chlorine, vinyl, isobutenyl, 2,2-diphenylvinyl, and carboxyl) and molecular weights (1000 to 4500 g/mol), initiated by monofunctional and aromatic bifunctional initiators were studied by atmospheric pressure photoionization mass spectrometry (APPI-MS) in both negative and positive ion modes. Consistent with previous findings, negative ion APPI-MS revealed end-group identities through the formation of PIB adducts with chloride ions formed in situ from a chlorinated solvent (e.g., CCl₄) in the presence of a dopant (toluene). In positive ion mode, considerable fragmentation of these PIB derivatives was observed, rendering end-group determinations very difficult. The M _n values obtained by APPI(−)-MS were considerably lower than those determined by SEC for PIB derivatives with M _n higher than 2000 g/mol. PIBs containing carboxyl termini can undergo collision-induced dissociation, yielding structurally important product ions. The resulting APPI-MS/MS intensities were found to reflect the “arm-length” distribution for PIBs with bifunctional aromatic moieties. In positive ion mode, [M+COCl]⁺ adducts were observed for PIBs with an aromatic initiator moiety. The origin of the COCl⁺ species is also discussed.     

Gas chromatography/mass spectrometry of polychlorinated biphenyls using atmospheric pressure chemical ionization and atmospheric pressure photoionization microchips

Gas chromatography (GC) and ion trap mass spectrometry (MS) were combined with microchip atmospheric pressure chemical ionization (microAPCI) and microchip atmospheric pressure photoionization (microAPPI) sources. Selected polychlorinated biphenyls (PCBs, IUPAC Nos. 28, 52, 101, 118, 138, 153 and 180) were analyzed by GC/microAPCI-MS and GC/microAPPI-MS to demonstrate the applicability of the miniaturized ion sources in negative ion mode analysis. The microAPCI and microAPPI methods were evaluated in respect of detection limit, linearity and repeatability. The detection limits for the PCB congeners were somewhat lower with microAPCI than with microAPPI, whereas microAPPI showed slightly wider linear range and better repeatability. With both methods, the best results were obtained for highly chlorinated or non-ortho-chlorinated PCBs, which possess the highest electron affinities. Finally, the suitability of the GC/microAPPI-MS method for the analysis of PCBs in environmental samples was demonstrated by analyzing soil extracts, and by comparing the results with those obtained by gas chromatography with electron capture detection (GC/ECD).     

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry for analysis of Dechloranes

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) was investigated as an instrumental method for the analysis of the halogenated norbornene flame retardants, Mirex, Dechloranes 602, 603, 604, and Dechlorane Plus (DP). The LC separation was optimized by screening a variety of stationary and mobile phases, resulting in a short LC separation time of 5 min. Different atmospheric pressure ionization approaches were examined including electrospray ionization, atmospheric pressure chemical ionization, and APPI, each with and without post-column addition. APPI without post-column addition was chosen for providing the best ionization response. The optimized LC/APPI-MS/MS approach resulted in instrument detection limits ranging between 25 and 50 pg. Good linearity was also achieved (up to 25.0 ng/μL; R >0.999). The method was applied to extracts of environmental samples including surface water, fish and sediments for screening purposes, and the results agreed well with those obtained by gas chromatography/mass spectrometry.     

Synthetic oligomer analysis using atmospheric pressure photoionization mass spectrometry at different photon energies

Atmospheric pressure photoionization (APPI) followed by mass spectrometric detection was used to ionize a variety of polymers: polyethylene glycol, polymethyl methacrylate, polystyrene, and polysiloxane. In most cases, whatever the polymer or the solvent used (dichloromethane, tetrahydrofuran, hexane, acetone or toluene), only negative ion mode produced intact ions such as chlorinated adducts, with no or few fragmentations, in contrast to the positive ion mode that frequently led to important in-source fragmentations. In addition, it was shown that optimal detection of polymer distributions require a fine tuning of other source parameters such as temperature and ion transfer voltage. Series of mass spectra were recorded in the negative mode, in various solvents (dichloromethane, tetrahydrofuran, hexane, toluene, and acetone), by varying the photon energy from 8 eV up to 10.6 eV using synchrotron radiation. To these solvents, addition of a classical APPI dopant (toluene or acetone) was not necessary. Courtesy of the synchrotron radiation, it was demonstrated that the photon energy required for an efficient ionization of the polymer was correlated to the ionization energy of the solvent. As commercial APPI sources typically use krypton lamps with energy fixed at 10 eV and 10.6 eV, the study of the ionization of polymers over a wavelength range allowed to confirm and refine the previously proposed ionization mechanisms. Moreover, the APPI source can efficiently be used as an interface between size exclusion chromatography or reverse phase liquid chromatography and MS for the study of synthetic oligomers. However, the photoionization at fixed wavelength of polymer standards with different molecular weights showed that it was difficult to obtain intact ionized oligomers with molecular weights above a few thousands.     

From fundamentals to applications: recent developments in atmospheric pressure photoionization mass spectrometry

Only five years after the first publication on atmospheric pressure photoionization (APPI), this technique has evolved rapidly as a very useful complement to established ionization techniques for liquid chromatography/mass spectrometry (LC/MS). This is reflected in a rapidly increasing number of publications in this field. On the one hand, thorough studies into the photoionization mechanism have provided deep insights into the roles and influences of the solvent, the dopant and other additives. On the other hand, a large number of new and attractive applications have recently been introduced. New instrumental developments have resulted in combined APPI/ESI (PAESI) and APPI/APCI sources and a microfabricated APPI source. In this review, the most important developments within the field are summarized, focusing in particular on the applications of the technique.     

Atmospheric pressure photoionization mass spectrometry of oligodeoxyribonucleotides

Small oligonucleotides (di- and trimers) were investigated by atmospheric pressure photoionization (APPI) with focus on the fragmentation mechanisms. The fragmentation patterns of these biomolecular ions have been monitored under dopant-assisted photoionization (DA-APPI) conditions. Our results reveal new aspects of the gas- phase chemistry of ions formed from such biomolecules. They illustrate that the reaction between low-energy electrons released from photoionization processes and di- and trinucleotides lies in dissociative electron attachment processes leading to phosphodiester bond cleavages and to the formation of numerous fragments in the ion source. The conditions of DA-APPI, which involve protic solvents and atmospheric pressure conditions, seem to be relevant for the study of radiation damages to biological molecules.     

Study of polybrominated diphenyl ethers using both positive and negative atmospheric pressure photoionization and tandem mass spectrometry

Atmospheric pressure photoionization (APPI) was assessed for the mass spectrometric analysis of polybromodiphenyl ethers (PBDEs) on the basis of a set of 17 standard compounds. Positive and negative ionization modes were both investigated. M(+.) ions were formed under positive ion conditions whereas the negative ion mode yielded [M-Br+O](-) ions. The behavior of these APPI-produced ions towards collisional activation was studied using an ion trap mass spectrometer. In positive ion mode, the loss of Br(2) was one of the major fragmentation pathways, and was favored for ortho-substituted PBDEs. Conversely, the loss of COBr(.) occurred only for non-ortho-substituted congeners. The collisional excitation of [M-Br+O](-) ions in the ion trap also led to the loss of Br(2), to the elimination of HBr, and to the formation of product ions by cleavage of the ether bond. The formation of para-quinone radical anions was observed for PBDEs ranging from penta- to hepta-congeners, whereas brominated aromatic carbanions were formed preferentially for the most brominated PBDEs studied in this work (hepta- or deca-BDEs). M(+.) ions did not undergo this fragmentation process.     

On the stability of the organic dication of the bisquaternary ammonium salt decamethoxinum under liquid secondary ion mass spectrometry

In the course of a liquid secondary ion mass spectrometric (SIMS) investigation on a bisquaternary ammonium antimicrobial agent, decamethoxinum, unusual pathways of fragmentation of the organic dication M2+ of this bisquaternary salt, with preservation of the doubly charged state of the fragments, were observed. To reveal the structural and electronic parameters of decamethoxinum, which are responsible for the stabilization of its organic dication in the gas phase, a comprehensive SIMS study using metastable decay, collision-induced dissociation and kinetic energy release techniques complemented by ab initio quantum chemical calculations was performed. Pathways of fragmentation of two main precursors originating from decamethoxinum-organic dication M2+ and its cluster with a Cl- counterion [M.Cl]+-and a number of their primary fragments were established and systematized. Differences in the pathways of fragmentation of M2+ and [M.Cl]+ were revealed: the main directions of [M.Cl]+ decay involve dequaternization similar to thermal degradation of this compound, while in M2+ fragmentation via loss of one and two terminal radicals with preservation of the doubly charged state of the fragments dominates over charge separation processes. It was shown that pairing of the dication with a Cl- anion does not preserve the complex from fragmentation via separation of two positively charged centers or neutralization (dequaternization) of one such center. At the same time the low abundance of M2+ in the SIMS spectra is to a larger extent controlled by a probability of M2+ association with an anion than by the decay of the dication per se.Quantum chemical calculations of the structural and electronic parameters of the decamethoxinum dication have revealed at least three features which can provide stabilization of the doubly charged state. Firstly, in the most energetically favorable stretch conformation the distance between the quaternary nitrogens (rN1-N2=1.39 nm) is relatively large. Secondly, an intramolecular solvation of quaternary groups by carbonyl oxygens of the adjacent groups of the dication occurs, which contribute to structural stabilization. Thirdly, an important feature of the electronic structure of the dication is the presence of a partial negative charge on the nitrogen atoms and smearing of a positive charge mainly over the hydrogens of alkyl groups attached to the quaternary nitrogens, which reduces the net repulsion between the quaternary groups. The possible influence of charge smearing on the kinetic energy released on the dication fragmentation is discussed.     

A dopant introduction device for atmospheric pressure photoionization with liquid chromatography/mass spectrometry

This technical note describes in detail the fabrication, operation and characterization of a pneumatically driven dopant introduction device, with a solvent reservoir capacity of 300 mL. Dopant flow rates and stability for this device are governed by the simple regulation of gas pressure rather than the progression of a stepper motor and syringe diameter, as is the case for typical infusion pumps. The device has the potential to provide days or even weeks of continuous, uninterrupted dopant flow at rates commonly adopted for atmospheric pressure photoionization (APPI) experiments without the need to replenish the dopant supply. Although not a refined instrumental design, this device was developed as an alternative cost-effective means of introducing stable dopant flow to an APPI source. The device was designed such that all components would be commercially available and easily procurable from common scientific part vendors. Figures and suggested part numbers are provided to allow those interested to fabricate similar devices to suit their individual experimental needs. Device characterization was performed while monitoring such factors as flow rate calibration, overall flow stability and reproducibility. In addition, a standard mixture of three polycyclic aromatic hydrocarbons was employed as a model sample for a typical reversed-phase liquid chromatography/atmospheric pressure photoionization mass spectrometry (LC/APPI-MS) application in order to demonstrate device performance.     

Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.     

Atmospheric pressure photoionization mass spectrometry of polyisobutylene derivatives

Atmospheric pressure photoionization mass spectrometric (APPI-MS) study on three types of polyisobutylene derivatives is reported. Two of the polyisobutylenes investigated were polyisobutylene with dihydroxy and diolefinic end-groups derived from aromatic moieties [dicumyl chloride, 1,4-bis(2-chloro-2-propyl)benzene], and the third contained no aromatic moieties with a monohydroxy end-group. All three polyisobutylene derivatives (PIBs) had an average molecular weight (M(n)) of approximately 2000 g/mol, with a polydispersity lower than 1.2. In the positive ion APPI mode, protonated PIB molecules were formed, but the molecular weights obtained were considerably lower than those expected, indicating fragmentation of the PIB chains. In the negative APPI mode, using solvents such as tetrahydrofuran and toluene as dopants, no signal was obtained. However, in chlorinated solvents, such as CCl(4), CHCl(3), and CH(2)Cl(2), in the presence of toluene dopant, PIB adducts with chloride ions were formed with relatively high signal intensity. In the case of CH(2)Cl(2), no dopant (toluene) was necessary to generate chlorinated adduct ions, albeit increasing the toluene concentration in the flow increased the PIB signal intensity. The effect of the toluene concentration on PIB signal intensity was studied and models that include (1) photoionization of toluene, (2) formation of chloride ions from the chlorinated solvents by dissociative electron capture, (3) formation of chlorinated adduct ions and charge recombination reactions between the toluene radical cation, (4) chloride ions, and (5) chlorinated adduct ions are proposed based on the experimental results.