Atmospheric pressure photoionization. 1. General properties for LC/MS

In this work, we describe the performance of an atmospheric pressure photoionization (APPI) source for sampling liquid flows. The results presented here primarily focus on the mechanism of direct photoionization (PI), as compared to the dopant mechanism of PI. Measured detection limits for direct APPI were comparable to atmospheric pressure chemical ionization (APCI; e.g., 1 pg for reserpine). The ion signal is linear up to 10 ng injected quantity, with a useful dynamic range exceeding 100 ng. Evidence is presented indicating that APPI achieves significantly better sensitivity than APCI at flow rates below 200 microL/min, making it a useful source for capillary liquid chromatography and capillary electrophoresis. Results are presented indicating that APPI is less susceptible to ion suppression and salt buffer effects than APCI and electrospray ionization (ESI). The principal benefit of APPI, as compared to other ionization sources, is in efficiently ionizing broad classes of nonpolar compounds. Thus, APPI is an important complement to ESI and APCI by expanding the range and classes of compounds that can be analyzed. In this paper, we also discuss the role of direct APPI vs PI-induced APCI using dopants.     

Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics

A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Gradient tandem mass spectrometry interfaced with ion mobility separation for the characterization of supramolecular architectures

Traveling wave ion mobility mass spectrometry (TWIM MS) was combined with gradient tandem mass spectrometry (gMS(2)) to deconvolute and characterize superimposed ions with different charges and shapes formed by electrospray ionization (ESI) of self-assembled, hexameric metallomacrocycles composed of terpyridine-based ligands and Cd(II) ions. ESI conditions were optimized to obtain intact hexameric cation assemblies in a low charge state (2+), in order to minimize overlapping fragments of the same mass-to-charge ratio. With TWIM MS, intact hexameric ions could be separated from remaining fragments and aggregates. Collisional activation of these hexameric ions at varying collision energies (gMS(2)), followed by TWIM separation, was then performed to resolve macrocyclic from linear hexameric species. Because of the different stabilities of these architectures, gMS(2) changes their relative amounts, which can be monitored individually after subsequent ion mobility separation. On the basis of this unique strategy, hexameric cyclic and linear isomers have been successfully resolved and identified. Complementary structural information was gained by the gMS(2) fragmentation pattern of the metallosupramolecules, acquired by collisionally activated dissociation after TWIM dispersion. TWIM MS interfaced with gMS(2) should be particularly valuable for the characterization of a variety of supramolecular polymers, which often contain isomeric architectures that yield overlapping fragments and aggregates upon ESI MS analysis.     

Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of "wrong-way around" ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple PKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative.     

APCI interface for LC- and SEC-MS analysis of synthetic polymers: advantages and limits

The main advantage of the APCI interface for the LC-MS analysis of synthetic polymers resides in its compatibility with the main chromatographic modes: reversed-phase liquid chromatography, normal-phase liquid chromatography, and size exclusion chromatography in organic phase, with the usual flow rates. Moreover, APCI can be used in positive or negative modes. Representative applications are described to highlight benefits and limitations of the LC-APCI-MS technique with the analysis of industrial polymers up to molecular masses of 5 kDa: polyethers; polysiloxanes; and copolymers of siloxanes. Results are discussed in regard to those obtained by more classical techniques: SEC and MALDI-MS. The use of an APCI interface in LC-MS and SEC-MS coupling applied to synthetic polymers is efficient up to 2000-4500 Da. The main drawback of the APCI interface is the in-source decomposition that is observed above m/z = 2000-3000 and can induce an underestimation of average molecular weights. However, APCI allows detection on a wide range of polarity of sample/solvent and appears to be complementary to ESI.     

Corona discharge ion mobility spectrometry with orthogonal acceleration time of flight mass spectrometry for monitoring of volatile organic compounds

We demonstrate the application of corona discharge ion mobility spectrometry with orthogonal acceleration time of flight mass spectrometry (CD IMS-oaTOF) for volatile organic compounds (VOCs) monitoring. Two-dimensional (2D) IMS-oaTOF spectra of VOCs were recorded in nearly real time. The corona discharge atmospheric pressure chemical ionization (APCI) source was operated in positive mode in nitrogen and air. The CD ion source generates in air H(3)O(+)(H(2)O)(n) and NO(+). The NO(+) offers additional possibility for selective ionization and for an increase of the sensitivity of monoaromatic compounds. In addition to H(3)O(+)(H(2)O)(n) and NO(+), we have carried out ionization of VOCs using acetone as dopant gas ((CH(3))(2)COH(+)). Sixteen model VOCs (tetrahydrofuran, butanol, n-propanol, iso-propano, acetone, methanol, ethanol, toluene, benzene, amomnia, dioxan, triethylamine, acetonitrile, formaldehyde, m-xylene, 2,2,2-trifluoroethylamine) were tested using these ionization techniques.     

Derivatization for stabilizing sialic acids in MALDI-MS

While matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is useful in oligosaccharide analysis, the sialic acid, or N-acetylneuraminic acid (NANA), moiety of an oligosaccharide is liable to dissociation in- or postsource during mass measurement. In this study, we tried to stabilize the moiety by amidation, as in the case of peptides (Sekiya, S.; Wada, Y. Tanaka, K. Anal. Chem. 2004, 76, 5894-5902), and found 4-(4,6-dimethoxy-1,3,5-triazin-2yl)-4-methylmorpholinium chloride to be a desirable condensing agent. Amidation stabilized the glycosidic bond with NANA and suppressed its preferential cleavage by in-source decay, postsource decay, or collision-induced dissociation. In addition, the suppressed dissociation considerably improved the yield of the B/Y type ions for structural analysis by MS/MS. These results demonstrate that amidation is an effective derivatization to reinforce the structural analysis of sialylated oligosaccharides by MALDI-MS. In addition, amidation with (15)N-labeled ammonium chloride decreases the mass shift from the acid to amide form to just 0.013, reducing the complexity of mass spectral interpretation and database searching.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

Comparison of electrospray,atmospheric pressure chemical ionization,and atmospheric pressure photoionization in the identification of apomorphine,dobutamine, and entacapone phase II metabolites in biological samples

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.     

Determination of bond dissociation energies using electrospray tandem mass spectrometry and a derived effective reaction path length approach

A new approach for calculating bond dissociation energies (BDEs) from ES-MS/MS measurements has been developed. The new method features a "derived effective reaction path length" that has been applied to measure BDEs of alkali metal (Li+) adducts and halide (Cl-) adducts of monoacylglycerol, 1,2-diacylglycerol, and 1,3-diacylglycerol lipids. Also studied were lithium-bound dimers of monoacylglycerols, 1,2-diacylglycerols, and 1,3-diacylglycerols. BDEs for the adducts and dimers of the lipids were derived from collision-induced dissociation experiments using a triple quadrupole mass spectrometer with electrospray as the ionization source. Mass spectral data were used to empirically derive a single-exponential growth equation that relates product cross section to collision energy. From these single-exponential equations, a general second-order polynomial was derived using a multivariate growth curve model that enables prediction of BDEs of unknown complexes. Mass spectral results were compared to computer-generated bond dissociation energies using Becke-style three-parameter density functional theory (B3LYP, employing the Lee-Yang-Parr correlation functional), with excellent agreement between experimental and theoretical energy values. The newly developed method is general in nature and can be used for the measurement of metal or halide ionic adduct bond dissociation energies and for the measurement of bond energies of noncovalent interactions such as dimer dissociation energies. The validity of the method has been rigorously established using a triple quadrupole, but it may also be applied to other mass spectrometers that allow user control of the collision cell potential.     

Pulsed hydrogen/deuterium exchange MS/MS for studying the relationship between noncovalent protein complexes in solution and in the gas phase after electrospray ionization

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.     

Analysis of saturated hydrocarbons by redox reaction with negative-ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

A novel technique was developed for characterization of saturated hydrocarbons. Linear alkanes were selectively oxidized to ketones by ruthenium ion catalyzed oxidation (RICO). Branched and cyclic alkanes were oxidized to alcohols and ketones. The ketones were then reduced to alcohols by lithium aluminum hydride (LiAlH(4)). The monohydric alcohols (O(1)) in the products obtained from the RICO and RICO-LiAlH(4) reduction reactions were characterized using negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for identification of iso-paraffins, acyclic paraffins and cyclic paraffins. Various model saturated compounds were used to determine the RICO reaction and ionization selectivity. The results from the FTICR MS analysis on the petroleum distillates derived saturated fraction were in agreement with those from field ionization gas chromatography time-of-flight mass spectrometry (FI GC-TOF MS) analysis. The technique was also used to characterize a petroleum vacuum residue (VR) derived saturates. The results showed that the saturated molecules in the VR contained up to 11 cyclic rings, and the maximum carbon number was up to 92.     

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.     

Capillary electrochromatography-mass spectrometry of zwitterionic surfactants

This work describes the on-line hyphenation of a packed capillary electrochromatography (CEC) column with an internally tapered tip coupled to electrospray ionization-mass spectrometry (ESI-MS) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) for the analysis of betaine-type amphoteric or zwitterionic surfactants (Zwittergent). A systematic investigation of the CEC separation and MS detection parameters comparing ESI and APCI is shown. First, a detailed and optimized manufacturing procedure for fabrication of the CEC-MS column with a reproducible internally tapered tip (7-9 microm) is presented. Next, the optimization of the separation parameters by varying the C(18) stationary-phase particle size (3 versus 1.5 microm), as well as mobile-phase composition including acetonitrile (ACN) volume fraction, ionic strength, and pH is described. The optimized separation is achieved using 3-microm C(18) packing with 75% ACN (v/v), 5 mM Tris at pH 8.0. Optimization for on-line CEC-ESI-MS detection is then done varying both the sheath liquid and spray chamber parameters while evaluating the use of random versus structured factorial table experimental designs. The more structured approach allows fundamental analysis of individual ESI-MS parameters while minimizing CEC and MS equilibration time between settings. A comparison of CEC-ESI-MS to CEC-APCI-MS using similar sheath and spray chamber conditions presents new insight for coupling of CEC to APCI-MS. The sheath liquid flow rate required to maintain adequate sensitivity is much higher in APCI source (50 microL/min) as compared to the ESI source (3 microL/min). The on-line mass spectra obtained in the full scan mode show that fragmentation in the two sources occurs at different positions on the Zwittergent molecules. For ESI-MS, the protonated molecular ion is always highest in abundance with minor fragmentation occurring due to the loss of the alkyl chain. In contrast, the APCI-MS spectra show that the highest abundant ion resulted by elimination of propane sulfonate from the Zwittergent molecule. A comparison of the sensitivity between the two sources in positive ionization SIM mode shows that CEC-ESI-MS provides an impressive limit of detection (LOD) of 5 ng/mL, which is at least 3 orders of magnitude lower than CEC-APCI-MS (LOD 100 microg/mL). Finally, the optimized CEC-MS methods comparing ESI and APCI are applied for separation and structural characterization of a real industrial zwittergent sample, Rewoteric AM CAS.     

Organic chloramine analysis and free chlorine quantification by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), together with tandem mass spectrometry (MSn), are used to study the mechanism of chlorination of amines and to develop a method for qualitative and quantitative determination of organic chloramines. Cyclohexylamine and 1,4-butanediamine (putrescine) are used as model compounds to investigate the mechanisms of the reactions between primary aliphatic amines and hypochlorous acid (aqueous Cl2). The chlorination products are identified and characterized by collision-induced dissociation (CID) and H/D exchange. Chlorination occurs by electrophilic addition of Cl+ and may be followed by HCl elimination, hydrolysis, or, in the case of diamines, amine elimination by intramolecular nucleophilic substitution. The relative rates of chlorination at amine and chloramine nitrogens are a function of pH and depend on the basicity of the amine. A novel method for active chlorine quantification using ESI or APCI mass spectrometry is suggested on the basis of the extent of chlorination of a sacrifical amine standard. This measurement has a limit of detection for N-chlorocyclohexylamine in the range of 0.1-10 microM, a linear dynamic range of 10(2)-10(3), and an accuracy of +/-10%, as determined for wastewater samples.     

Breaking the pumping speed barrier in mass spectrometry: discontinuous atmospheric pressure interface

The performance of mass spectrometers with limited pumping capacity is shown to be improved through use of a discontinuous atmospheric pressure interface (DAPI). A proof-of-concept DAPI interface was designed and characterized using a miniature rectilinear ion trap mass spectrometer. The interface consists of a simple capillary directly connecting the atmospheric pressure ion source to the vacuum mass analyzer region; it has no ion optical elements and no differential pumping stages. Gases carrying ionized analytes were pulsed into the mass analyzer for short periods at high flow rates rather than being continuously introduced at lower flow rates; this procedure maximized ion transfer. The use of DAPI provides a simple solution to the problem of coupling an atmospheric pressure ionization source to a miniature instrument with limited pumping capacity. Data were recorded using various atmospheric pressure ionization sources, including electrospray ionization (ESI), nano-ESI, atmospheric pressure chemical ionization (APCI), and desorption electrospray ionization (DESI) sources. The interface was opened briefly for ion introduction during each scan. With the use of the 18 W pumping system of the Mini 10, limits of detection in the low part-per-billion levels were achieved and unit resolution mass spectra were recorded.     

Comparison of atmospheric pressure photoionization, atmospheric pressure chemical ionization, and electrospray ionization mass spectrometry for analysis of lipids

In this work, we compare the quantitative accuracy and sensitivity of analyzing lipids by atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) LC/MS. The target analytes include free fatty acids and their esters, monoglyceride, diglyceride, and triglyceride. The results demonstrate the benefits of using LC/APPI-MS for lipid analysis. Analyses were performed on a Waters ZQ LC/MS. Normal-phase solvent systems were used due to low solubility of these compounds in aqueous reversed-phase solvent systems. By comparison, APPI offers lower detection limits, generally highest signal intensities, and the highest S/N ratio. APPI is 2-4 times more sensitive than APCI and much more sensitive than ESI without mobile-phase modifiers. APPI and APCI offer comparable linear range (i.e., 4-5 decades). ESI sensitivity is dramatically enhanced by use of mobile phase modifiers (i.e., ammonium formate or sodium acetate); however, these ESI adduct signals are less stable and either are nonlinear or have dramatically reduced linear ranges. Analysis of fish oils by APPI shows significantly enhanced target analyte intensities in comparison with APCI and ESI.     

Effect of experimental parameters on the ESI FT-ICR mass spectrum of fulvic acid

Fulvic acid (FA) is a heterogeneous mixture of organic macromolecules found in the waters, soils, and sediments of the earth's surface. The ability of electrospray ionization (ESI) to effectively transfer large ions from the solution phase to the gas phase and the coupling of ESI to the high-mass-resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provide a potential method for the mass spectrometric analysis of FA. Positive- and negative-ion ESI FT-ICR MS analyses of four reference International Humic Substances Society FAs were performed. The spray solution composition was found to have a dramatic effect on the ion distributions, with high-mass aggregates (m/z approximately 2000-4000) being formed in less polar spray solutions. Positive-ion spectra for each FA obtained under optimum conditions resulted in number-average molecular weights ranging from 1700 to 1900. The mass spectra were extremely complex, with ion distributions on the order of m/z approximately 500-3000. The presence of more than one ion at each nominal mass was routinely observed. Negative-ion ESI analysis of the FA samples resulted in the observation of multiply charged ions whose distributions could be affected by the acidification of the spray solution. Solution parameters which have been reported to affect molecular weight distributions of FA such as pH, ionic strength, and concentration of multivalent cations were found to have little or no effect on the observed m/z distributions.     

Reactive-electrospray-assisted laser desorption/ionization for characterization of peptides and proteins

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS ( n ) to protein analysis and show that ELDI is capable of multistage MS to MS (4) for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce "reactive-ELDI", which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.