On-line capillary electrophoresis-mass spectrometry using dopant-assisted atmospheric pressure photoionization: setup and system performance

The on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) via atmospheric pressure photoionization (APPI) is demonstrated. To achieve CE-APPI-MS, an adapted coaxial sheath-flow interface was combined with an ion-trap mass spectrometer equipped with an APPI source originally designed for liquid chromatography-MS. Effective photoionization of test compounds was accomplished after optimization of several interface and MS parameters, and of the composition and flow rate of the sheath liquid. Further enhancement of the ionization efficiency could be achieved by adding a dopant, such as acetone or toluene, to the sheath liquid to aid indirect ionization. Acetone significantly increased the ionization of the polar test compounds by proton transfer, while toluene was more useful for the enhanced formation of molecular ions from nonpolar compounds. The effect of several common CE background electrolytes (BGEs) on the APPI-MS response of the analytes was also studied. It appeared that in contrast with electrospray ionization, nonvolatile BGEs do not cause suppression of analyte signals using APPI. Therefore, in CE-APPI-MS, a variety of buffers can be chosen, which obviously is a great advantage during method development. Remarkably, also sodium dodecyl sulfate (SDS) did not affect the photoionization of the test compounds, indicating a strong potential of APPI for the on-line coupling of micellar electrokinetic chromatography (MEKC) and MS.     

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.     

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.     

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 for coupling liquid-chromatography to mass spectrometry: A review

This review presents the state-of-the-art techniques that couple liquid chromatography (LC) and mass spectrometry (MS) via atmospheric pressure photoionization (APPI). The different ionization mechanisms are discussed as well as the influence of the mobile phase composition, the nature of the dopant, etc. A comparison with other ionization sources, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), is reported, and the combination of APPI with these sources is also discussed. Several applications, covering the time period of 2005-2008, for the analysis of drugs, lipids, natural compounds, pesticides, synthetic organics, petroleum derivatives, and other substances are presented.     

Determination of polymer additives by liquid chromatography coupled with mass spectrometry. A comparison of atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI)

A method for the determination of polymer additives like antioxidants, UV absorbers and processing stabilizers using liquid chromatography (LC) coupled with atmospheric pressure photoionization mass spectrometry (APPI-MS) is presented. Ion source parameters were optimized regarding temperatures, gas flow rates, and voltages applied. Detection limits were determined using APPI with or without dopant and were compared with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Differences between APPI, ESI and APCI are pointed out and the effect of the dopant toluene and acetone is discussed. The optimized method yielded detection limits between 0.001 mg L⁻¹ and 0.022 mg L⁻¹ for 15 different analytes. Linear calibration plots could be obtained for all solutes over a wide concentration range showing satisfying repeatability with standard deviations of peak areas between 3.4% and 7.6%. The results indicate that the developed method can be regarded as suitable for the quantitative determination of polymer additives even at low concentration levels.     

Basic rules for the interpretation of atmospheric pressure ionization mass spectra of small molecules

This review summarizes the basic rules for the interpretation of atmospheric pressure ionization (API) mass spectra of small molecules written with the style primarily intended for beginners and low-experienced researchers with the mass spectra interpretation. The first and basic step in any interpretation of mass spectra is always the determination of molecular weight, which is relatively easy in case of soft ionization techniques due to the limited extend of fragmentation and the prevailing presence of (de)protonated molecules in the full scan mass spectra. These [M+H]⁺ and [M−H]⁻ ions are often accompanied by low abundant molecular adducts, which can be used as the supplementary information for the unambiguous determination of molecular weights. In certain cases, adduct ions may dominate the spectra. The subsequent interpretation of full scan and tandem mass spectra is more complicated due to a high number of possible functional groups, structural subunits and their combinations resulting in numerous competitive fragmentation pathways. Typical neutral losses and the effect of individual functional groups on the fragmentation are discussed in detail and illustrated with selected examples. Modern mass analyzers have powerful features for the structural elucidation, for example high resolving power, high mass accuracy, multistage tandem mass spectrometry, dedicated softwares for the interpretation of mass spectra and prediction of their fragmentation. Background information on differences among individual ionization techniques suitable for the HPLC–MS coupling and basic types of mass analyzers with consequences for the data interpretation is briefly discussed as well. Selected examples illustrate that the right optimization of chromatographic separation and the use of other than mass spectrometric detectors can bring valuable complementary information.     

State-of-the-art in atmospheric pressure photoionization for LC/MS

This review presents our perspective on the state-of-the-art of atmospheric pressure photoionization (APPI) for LC/MS. Its focus is on APPI's capabilities and how to utilize them fully. The introduction includes a brief recounting of the history of APPI's development, as well as a summary of its operating principles and current position in the field. The primary ionization mechanisms in APPI are then addressed, including direct analyte photoionization (PI), dopant/solvent PI, and thermospray. Next a summary of the ion-molecule reaction pathways available for analyte ionization is presented, along with the conditions required for activating them. APPI's performance characteristics are then examined. In effect, this review is an interim report on progress made since Rafaelli and Saba concluded that “The ability…to direct the preferential ion formation towards one particular type…can be extremely useful for qualitative and quantitative determinations. For this purpose, a better insight in the processes involved in the ionization step is strongly needed” [A. Raffaelli, A. Saba, Mass Spectrom. Rev. 22 (2003) 318]. In the conclusion, we focus on areas of APPI technology identified as being either unoptimized or largely unexplored, and having the potential to be improved upon—the crux being that with further research and development improvements in the performance, capabilities, and ease-of-use of APPI may reasonably be anticipated.     

Ultrasonic nebulization extraction/low pressure photoionization mass spectrometry for direct analysis of chemicals in matrices

A novel ultrasonic nebulization extraction/low-pressure photoionization (UNE-LPPI) system has been designed and employed for the rapid mass spectrometric analysis of chemicals in matrices. An ultrasonic nebulizer was used to extract the chemicals in solid sample and nebulize the solvent in the nebulization cell. Aerosols formed by ultrasonic were evaporated by passing through a transferring tube, and desolvated chemicals were ionized by the emitted light (10.6 eV) from a Krypton discharge lamp at low pressure (∼68 Pa). First, a series of semi/non-volatile compounds with different polarities, such as polycyclic aromatic hydrocarbons (PAHs), amino acids, dipeptides, drugs, nucleic acids, alkaloids, and steroids were used to test the system. Then, the quantification capability of UNE-LPPI was checked with: 1) pure chemicals, such as 9,10-phenanthrenequinone and 1,4-naphthoquinone dissolved in solvent; 2) soil powder spiked with different amounts of phenanthrene and pyrene. For pure chemicals, the correlation coefficient (R²) for the standard curve of 9,10-phenanthrenequinone in the range of 3 ng–20 μg mL⁻¹ was 0.9922, and the measured limits of detection (LOD) was 1 ng ml⁻¹. In the case of soil powder, linear relationships for phenanthrene and pyrene from 10 to 400 ng mg⁻¹ were obtained with correlation coefficients of 0.9889 and 0.9893, respectively. At last, the feasibility of UNE-LPPI for the detection of chemicals in real matrices such as tablets and biological tissues (tea, Citrus aurantium peel and sage (Salvia officinalis) leaf) were successfully demonstrated.     

The detection and mapping of the spatial distribution of insect defense compounds by desorption atmospheric pressure photoionization Orbitrap mass spectrometry

Many insects use chemicals synthesized in exocrine glands and stored in reservoirs to protect themselves. Two chemically defended insects were used as models for the development of a new rapid analytical method based on desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). The distribution of defensive chemicals on the insect body surface was studied. Since these chemicals are predominantly nonpolar, DAPPI was a suitable analytical method. Repeatability of DAPPI-MS signals and effects related to non-planarity and roughness of samples were investigated using acrylic sheets uniformly covered with an analyte. After that, analytical figures of merit of the technique were determined. The spatial distribution of (E)-1-nitropentadec-1-ene, a toxic nitro compound synthesized by soldiers of the termite Prorhinotermes simplex, was investigated. Then, the spatial distribution of the unsaturated aldehydes (E)-hex-2-enal, (E)-4-oxohex-2-enal, (E)-oct-2-enal, (E,E)-deca-2,4-dienal and (E)-dec-2-enal was monitored in the stink bug Graphosoma lineatum. Chemicals present on the body surface were scanned along the median line of the insect from the head to the abdomen and vice versa, employing either the MS or MS² mode. In this fast and simple way, the opening of the frontal gland on the frons of termite soldiers and the position of the frontal gland reservoir, extending deep into the abdominal cavity, were localized. In the stink bug, the opening of the metathoracic scent glands (ostiole) on the ventral side of the thorax as well as the gland reservoir in the median position under the ventral surface of the anterior abdomen were detected and localized. The developed method has future prospects in routine laboratory use in life sciences.     

Quantitation of hindered amine light stabilizers in plastic materials by high performance liquid chromatography and mass spectrometric detection using electrospray ionization and atmospheric pressure photoionization

In this work new high performance liquid chromatographic methods in combination with mass spectrometry have been developed for the quantitation of hindered amine light stabilizers (HALS) which are commonly used as monomeric and oligomeric species for stabilization of plastic materials. These analytes are difficult to separate under traditional reversed phase conditions. In the present study new silica-based pH stable reversed phases that had become available recently were investigated for HALS analysis, and turned out to be well suited employing mobile phases at a pH of around 11 adjusted by addition of ammonia. Detection was done by mass spectrometry employing both time-of-flight and triple quadrupole mass analyzers. The performance of electrospray ionization (ESI) as well as atmospheric pressure photoionization (APPI) was investigated and compared. Despite the high pH of the mobile phase, an excellent ionization could be obtained in the positive ion mode. ESI provided slightly lower limits of quantitation (on average a factor of 2) in comparison with APPI. The method allowed the quantitation of a range of different HALS down to 0.05–0.005% (depending on the HALS) in polymeric materials. Sample preparation consisted in dissolving the sample in toluene and precipitation of the polymer with acetone. The procedure can be routinely applied to aging tests of plastic materials in order to predict the lifetime of plastic components.     

Mixed-mode and reversed-phase liquid chromatography-tandem mass spectrometry methodologies to study composition and base hydrolysis of polysorbate 20 and 80

Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate. The results indicated POE sorbitan was present from 16.0 to 27.6 and 11.1 to 14.5% (w/w) in polysorbate 20 and 80, respectively. The second HPLC method used a reversed-phase stationary phase (Zorbax SB-300 C(8)) with a shallow gradient to separate, identify, and quantify the multiple ester species present in polysorbate. For all lots of polysorbate 20 analyzed, only 18-23% of the material was the expected structure, polyoxyethylenesorbitan monolaurate. Up to 40% and 70% (w/w) di- and triesters were found in polysorbate 20 and polysorbate 80 respectively. Likewise, polyoxyethylenesorbitan monooleate accounted for only 20% of polysorbate 80. A variability of 3-5% was observed for each ester species between multiple lots of polysorbate 20. The reversed-phase method was then used to determine the rate of hydrolysis for each polyoxyethylene sorbitan ester of polysorbate 20 in basic solution at room temperature. Increasing rates of hydrolysis were observed with decreasing aliphatic chain lengths in polysorbate 20.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Atmospheric pressure photoionization-mass spectrometry and atmospheric pressure chemical ionization-mass spectrometry of neurotransmitters

A group of five neurotransmitters with different properties was analyzed using atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The sensitivity of the techniques for the analytes was tested in six solvents and in positive and negative ion modes. APPI was found to be superior in sensitivity for all the compounds in both positive and negative ion modes. In positive ion mode, water/methanol/formic acid was found to be the best solvent, whereas in negative ion mode, water/methanol/ammonium hydroxide performed best. Detection limits using APPI were between 2.5-250 fmol, depending on the compound. The sensitivity was best for the neurosteroids dehydroepiandrosterone and beta-estradiol, and acetylcholine (LOD 2.5-10 fmol).     

Atmospheric pressure photoionization mass spectrometry for analysis of fatty acid and acylglycerol lipids

In this work, we optimize parameters and conditions for analysis of fatty acid ester and acylglycerol lipids by atmospheric pressure photoionization-mass spectrometry (APPI-MS). The investigated parameters include atmospheric pressure chemical ionization (APCI) nebulizer/vaporizer physical orientation and APPI lamp face position, solvent selections, mobile phase compositions and flow rates, cone voltages and probe temperatures. APPI sensitivity is found to be highly dependent on mobile phase compositions. Normal phase solvents offer much higher sensitivity and better peak shape than reversed phase for nonpolar lipids. Hexane and isooctane are found to be two solvents generating highest S/N for eicosapentaenoic acid (EPA) methyl ester. The effects of mobile phase flow rates on sensitivity are found to be target analytes and target ions specific. However, the flow rate changes do not significantly affect the sensitivity of three out of four tested analytes under normal phase conditions over tested flow rates of 50-500muL/min. Cone voltage is found to be one of key parameters affecting sensitivity. Optimum probe temperature is found to be more dependent on mobile phase compositions than on the specific target analytes. Aqueous reversed-phase mobile phase requires higher probe temperature than normal phase for better sensitivity. More volatile mobile phase solvents require lower probe temperature for analyte desolvation. APPI offers four to five decades of linear ranges under normal phase condition. Full scan mass spectra of individual lipid standards, custom lipid mixtures and natural fish oil show that APPI spectra are clean and very easy to interpret. APPI also gives stable, reproducible peak responses with good peak shape. Limits of detection (LODs) by FIA (S/N=3) are estimated to be 12pg for EPA methyl ester and monoarachidin, 19pg for diarachidin and 7pg for trielaidin. LODs on-column are estimated to be 94pg for EPA methyl ester, 90pg for monoarachidin and diarachidin and 24pg for trielaidin.     

Atmospheric pressure ion lens extends the stable operational region of an electrospray ion source for capillary electrophoresis-mass spectrometry

An atmospheric ion lens incorporated into an electrospray ion source for capillary electrophoresis-mass spectrometry (CE-MS) is found to extend the stable operational regions for both flow rates and electrospray ionization (ESI) voltages. The stable operating conditions for the ESI source with and without the ion lens were characterized. The results showed that the stable operation region was widest when the voltage difference between the sprayer and the ion lens ranges from 2.6 to 2.8 kV, and under these condition, the CE-MS interface can be adapted to a broader range of electroosmotic and modifier flow rates. Modeling of the electric field in the electrospray ion source with the ion lens suggests that the extension of the stable region is attributed to the flatter equipotential surfaces around the sprayer tip and higher electric field strengths in the rest of the interface region.     

Improvement using chemometrics in ion mobility coupled to mass spectrometry as a tool for mass spectrometry fragmentation studies: Flavonoid aglycone cases

A chemometric method for the treatment of ion mobility coupled to mass spectrometry (IMS/MS) data is proposed as a complementary tool for obtaining experimental evidence for the study of MS fragmentations, which can provide a direct and automatable methodology for characterising ionic series and the hierarchy of all product ions of an MS spectrum.     

Atmospheric pressure chemical ionization of explosives induced by soft X‐radiation in ion mobility spectrometry: mass spectrometric investigation of the ionization reactions of drift gasses,dopants and alkyl nitrates

A promising replacement for the radioactive sources commonly encountered in ion mobility spectrometers is a miniaturized, energy‐efficient photoionization source that produce the reactant ions via soft X‐radiation (2.8 keV). In order to successfully apply the photoionization source, it is imperative to know the spectrum of reactant ions and the subsequent ionization reactions leading to the detection of analytes. To that end, an ionization chamber based on the photoionization source that reproduces the ionization processes in the ion mobility spectrometer and facilitates efficient transfer of the product ions into a mass spectrometer was developed. Photoionization of pure gasses and gas mixtures containing air, N₂, CO₂ and N₂O and the dopant CH₂Cl₂ is discussed. The main product ions of photoionization are identified and compared with the spectrum of reactant ions formed by radioactive and corona discharge sources on the basis of literature data. The results suggest that photoionization by soft X‐radiation in the negative mode is more selective than the other sources. In air, adduct ions of O₂^– with H₂O and CO₂ were exclusively detected. Traces of CO₂ impact the formation of adduct ions of O₂^– and Cl^– (upon addition of dopant) and are capable of suppressing them almost completely at high CO₂ concentrations. Additionally, the ionization products of four alkyl nitrates (ethylene glycol dinitrate, nitroglycerin, erythritol tetranitrate and pentaerythritol tetranitrate) formed by atmospheric pressure chemical ionization induced by X‐ray photoionization in different gasses (air, N₂ and N₂O) and dopants (CH₂Cl₂, C₂H₅Br and CH₃I) are investigated. The experimental studies are complemented by density functional theory calculations of the most important adduct ions of the alkyl nitrates (M) used for their spectrometric identification. In addition to the adduct ions [M + NO₃]^– and [M + Cl]^–, adduct ions such as [M + N₂O₂]^–, [M + Br]^– and [M + I]^– were detected, and their gas‐phase structures and energetics are investigated by density functional theory calculations. Copyright © 2016 John Wiley & Sons, Ltd.