Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.     

Unusual Analyte-Matrix Adduct Ions and Mechanism of Their Formation in MALDI TOF MS of Benzene-1,3,5-Tricarboxamide and Urea Compounds

Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks were always recorded when common MALDI matrices such as 4-hydroxy-α-cyanocinnamic acid (CHCA) were used. These compounds are mainly those with a benzene-1,3,5-tricarboxamide (BTA) or urea moiety, which are important building blocks to make new functional supramolecular materials. The possible mechanism of the adduct formation was investigated. A shared feature of the compounds studied is that they can form intermolecular hydrogen bonding with matrices like CHCA. The intermolecular hydrogen bonding will make the association between analyte ions and matrix molecules stronger. As a result, the analyte ions and matrix molecules in MALDI clusters will become more difficult to be separated from each other. Furthermore, it was found that analyte ions were mainly adducted with matrix salts, which is probably due to the much lower volatility of the salts compared with that of their corresponding matrix acids. It seems that the analyte-matrix adduct formation for our compounds are caused by the incomplete evaporation of matrix molecules from the MALDI clusters because of the combined effects of enhanced intermolecular interaction between analyte-matrix and of the low volatility of matrix salts. Based on these findings, strategies to suppress the analyte-matrix adduction are briefly discussed. In return, the positive results of using these strategies support the proposed mechanism of the analyte-matrix adduct formation.

Influence of the laser intensity and spot size on the desorption of molecules and ions in matrix-assisted laser desorption/ionization with a uniform beam profile

The dependence of the number of desorbed particles on laser fluence has been investigated for matrix-assisted laser desorption/ionization (MALDI) of analyte and matrix ions as well as for (photoionized) neutral matrix molecules using a homogeneous “flat-top” laser profile. Laser spot diameters ranging from 10 to 200 μm in size have been used. 2,5-Dihydroxybenzoic acid (DHB) and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) have been tested as matrices. The threshold (for ion detection) is higher and the dependence of the ion signal upon higher-than-threshold fluences is stronger for directly desorbed ions than for photoionized neutral molecules. Directly desorbed analyte ions exhibit the same dependence on fluence as the matrix ions with only minor differences between the two matrices tested, so both have approximately the same detection threshold. For both ions and photoionized neutral molecules, the fluence threshold increases with decreasing spot size while the slope of the intensity/fluence curves decreases. A quasi-thermal, sublimation/desportion model was found to describe the experimental results with excellent precision. For a complete explanation, non-equilibrium effects had to be taken into account.     

Clustering of pentacene and functionalized pentacene ions in a matrix-assisted laser desorption/ionization orthogonal TOF mass spectrometer

A high-performance orthogonal time-of flight (TOF) mass spectrometer, in combination with the matrix assisted laser desorption/ionization (MALDI) source operating at elevated pressure (∼1 torr in N₂), was used to perform MALDI-TOF analyses of pentacene and some of its derivatives with and without an added matrix. These molecules are among the most interesting semiconductor materials for organic thin film transistor applications (OTFT). The observation of ion-molecule reactions between “cold” analyte ions and neutral analyte molecules in the gas phase has provided some insight into the mechanism of pentacene cluster formation and its functionalized derivatives. Furthermore, some of the matrices employed to assist the desorption/ionization process of these compounds were observed to influence the outcome via ion-molecule reactions of analyte ions and matrix molecules in the gas phase. The stability and reactivity of the compounds and their clusters in the MALDI plume during gas-phase expansion were evaluated; possible structures of the resulting clusters are discussed. The MALDI-TOF technique was also helpful in distinguishing between two isomeric forms of bis-[(triisopropylsilyl)-ethynyl]-pentacene.     

Adduct-ion formation in trapped ion mobility spectrometry as a potential tool for studying molecular structures and conformations

Recent developments in the field of ion mobility spectrometry provide new possibilities to explore and understand gas-phase ion chemistry. In this study, hyphenated trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was applied to investigate analyte ion mobility as function of adduct ion formation for twelve pharmaceutically relevant molecules, and for tetrahydrocannabinol (THC) and its isomer cannabidiol (CBD). Samples were introduced by direct infusion and ions were generated with positive electrospray ionization (ESI+) observing protonated and sodiated ions. Measurements were performed with and without addition of cesium-, lithium-, silver- and sodium ions to the samples. For the tested compounds, metal adduct ions with the same m/z but with different mobility and collision cross section (CCSs) were observed, indicating different molecular conformations. Formation of analyte dimers was also observed, which could be associated with molecular geometry of the compounds. By optimizing the range and speed of the electric field gradient and ramp, respectively, the separation of THC and CBD was achieved by employing the adduct formation. This study demonstrates that the favorable resolution of TIMS combined with the ability to detect weakly bound counter ions is a valuable means for rapid detection, separation and structural assignment of molecular isomers and analyte conformations.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results.     

Injection of reagent ions into the selvedge region in fast-atom bombardment mass spectrometry

A divided probe that incorporates a potassium aluminosilicate glass target and an analyte/glycerol matrix target, spatially separated, was used to inject potassium ions (K⁺) into the high-pressure “selvedge” region formed above the analyte/glycerol matrix target during fast-atom bombardment (FAB); [M+K]⁺ adduct ions that represent the types of gas-phase neutral molecules present in the selvedge region are observed. Computer modeling assisted in designing the divided target and an additional ion optical element for the FAB ion source to optimize interactions between K⁺ ions and the desorbed neutral molecules. The capability of injecting K⁺ ions into the FAB experiment has utility in both mechanistic studies and analyses. Experimental results here are consistent with a model for the desorption/ionization processes in FAB in which some types of neutral analyte molecules are desorbed intact and are subsequently protonated by glycerol chemical ionization. Unstable protonated molecules undergo unimolecular decomposition to yield observed fragment ions. The use of K⁺ cationization of analytes for molecular weight confirmation is demonstrated, as well as its utility in FAB experiments in which mixtures are encountered.     

On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra

A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a delayed extraction (DE) technique, the flight time of an ion changes linearly with extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ～ 300 versus ～ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with delayed extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.     

Online Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with In situ Mixing

Online matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is reported with in situ matrix/analyte aerosol mixing. The analyte and matrix were dissolved in separate solvents and pneumatically atomized into particles. Continuous flows of analyte particles and matrix droplets in nitrogen were mixed in a tee. The resulting particles were sampled using a nozzle, focused into a beam with an aerodynamic lens system, and vertically deposited continuously on a movable target. The matrix/analyte mixture was desorbed/ionized using a 266 nanometers pulsed laser at an incident angle of 45°. The nascent ions were analyzed using reflectron TOF MS. The performance of online MALDI-TOF MS was evaluated by the analysis of palmityl palmitate with the lithium salt of 2, 4-dihydroxybenzoic acid as the matrix. Strong and stable MALDI signals of palmityl palmitate were obtained. The matrix solvent mixture and the analyte concentration were optimized and the results demonstrate the development of an alternative for online MALDI analysis.     

A novel strategy for MALDI-TOF MS analysis of small molecules

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) does not work efficiently on small molecules (usually with molecular weight below 500 Da) because of the interference of matrix-related peaks in low m/z region. The previous methods developed for this problem focused on reducing the peaks caused by the traditional matrices. Here, we report a novel strategy to analyze small molecules in a high and interference-free mass range by using metal-phthalocyanines (MPcs) as matrices which should be capable of forming matrix-analyte adducts. The mass of the target analyte was calculated by subtracting the mass of MPc from the mass of the MPc-analyte adduct. MPcs were also detectable and could serve as internal standards. Various MPcs with aromatic or aliphatic groups and different metal centers were then synthesized and explored. Aluminum-phthalocyanines (AlPcs), gallium-phthalocyanines (GaPcs), and indium-phthalocyanines (InPcs) were efficient matrices to form MPc-analyte adducts in either the positive or negative ion mode. The detection limits varied from 17 to 75 fmol, depending on analyte types. The mechanism of adducts formation was also proposed. Collectively, our strategy provides a novel and efficient way to analyze small molecules by MALDI-TOF MS.     

Reduction of in-source collision-induced dissociation and thermolysis of sulopenem prodrugs for quantitative liquid chromatography/electrospray ionization mass spectrometric analysis by promoting sodium adduct formation

Six chromatographically resolved sulopenem prodrugs were monitored for their potential to undergo both in-source collision-induced dissociation (CID) and thermolysis. Initial Q1 scans for each prodrug revealed the formation of intense [Prodrug2 + H]+, [Prodrug2 + Na]+, [Prodrug + Na]+, and [Sulopenem + Na]+ ions. Non-adduct-associated sulopenem ([Sulopenem + H]+) along with several additional lower mass ions were also observed. Product ion scans of [Prodrug3 + Na]+ showed the retention of the sodium adduct in the collision cell continuing down to opening of the beta-lactam ring. In-source CID and temperature experiments were conducted under chromatographic conditions while monitoring several of the latter ion transitions (i.e., adducts, dimers and degradants/fragments) for a given prodrug. The resulting ion profiles indicated the regions of greatest stability for temperature and declustering potential (DP) that provided the highest signal intensity for each prodrug and minimized in-source degradation. The heightened stability of adduct ions, relative to their appropriate counterpart (i.e., dimer to dimer adduct and prodrug to prodrug adduct ions), was observed under elevated temperature and DP conditions. The addition of 100 microM sodium to the mobile phase further enhanced the formation of these more stable adduct ions, yielding an optimal [Prodrug + Na]+ ion signal at temperatures from 400 to 600 degrees C. A clinical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for sulopenem prodrug PF-04064900 in buffered whole blood was successfully validated using sodium-fortified mobile phase and the [PF-04064900 + Na]+ ion for quantitation. A conservative five-fold increase in sensitivity from previously validated preclinical assays using the [PF-04064900 + H]+ precursor ion was achieved.     

Formation and fate of ion pairs during MALDI analysis: anion adduct generation as an indicative tool to determine ionization processes

Stimulated by recent experiments, which verified the preservation of the analyte solution charge state upon incorporation in the host matrix crystals, investigations are reported focusing on the role of analyte and counter ions in the matrix-assisted laser desorption/ionization (MALDI) process. These counter ions are only visible in the MALDI mass spectra under certain conditions, i.e., if inter-ionic proton transfer followed by evaporation of the neutrals is prevented, as in the case of metal cations. However, ion pairs can also survive the MALDI process if anions of very low gas phase basicities are used. By this means the intermediates of ion production in MALDI can be visualized. Depending on the amount of energy transfer to the analyte, which is mainly controlled by the matrix, different grades of adduct generation are observed. The analyte-, matrix- and polarity-dependant adduct distribution substantiates the hypothesis that multi-ion pairs are incorporated in the MALDI crystals and that ionization is essentially accomplished by charge separation processes. Moreover, the adduct distribution--and most probably also the charge separation efficiency--was found to be caused mainly by competition of different anionic species for coordination at the positively charged analyte sites. Furthermore, the results point to a less efficient charge separation with increasing number of ion pairs, which might be one major reason that mainly singly charged ions are obtained with MALDI.     

Negative fast atom bombardment ionization of aromatic sulfonic acids: unusual sample-matrix interaction

The most intense ion(s) in negative ion fast atom bombardment (FAB) mass spectra of 2- and 4-benzaldehyde sulfonic acid (BSA) in glycerol or 3-nitrobenzyl alcohol matrix corresponds to a covalent association of the analyte with one or two matrix molecules accompanied by the elimination of a molecule of water. The molecular ion [M - H](-), however, is of low abundance. The identity of the resulting ions [M + nA - H(2)O - H](-) (where M is the analyte and A is the matrix) was confirmed by exact mass measurement using the peak matching technique. These covalent matrix-analyte complexes were not observed when the sulfonic acid functionality in BSA was substituted with COOH, NO(2), and OH or when the sulfonic acid was in salt form. These observations indicate that the free sulfonic acid group in BSA is responsible for the covalent adduct formation. To our knowledge, analyte-matrix covalent association in negative ion FAB spectra of BSA has not been reported previously.     

Comparison of dielectric barrier discharge, atmospheric pressure radiofrequency-driven glow discharge and direct analysis in real time sources for ambient mass spectrometry of acetaminophen

Three plasma-based ambient pressure ion sources were investigated; laboratory constructed dielectric barrier and rf glow discharges, as well as a commercial corona discharge (DART source). All were used to desorb and ionize a model analyte, providing sampling techniques for ambient mass spectrometry (MS). Experimental parameters were optimized to achive highest signal for acetaminophen as the analyte. Insight into the mechanisms of analyte desorption and ionization was obtained by means of emission spectrometry and ion current measurements. Desorption and ionization mechanisms for this analyte appear to be identical for all three plasma sources. Emission spectra differ only in the intensities of various lines and bands. Desorption of solid analyte requires transfer of thermal energy from the plasma source to sample surface, in the absence of which complete loss of MS response occurs. For acetaminophen, helium was the best plasma gas, providing 100- to 1000-fold higher analyte response than with argon or nitrogen. The same trend was also evident with background ions (protonated water clusters). MS analyte signal intensity correlates with the ion density (expressed as ion current) in the plasma plume and with emission intensity from excited state species in the plasma. These observations support an ionization process which occurs via proton transfer from protonated water clusters to analyte molecules.     

If the ionization mechanism in fast-atom bombardment involves ion/molecule reactions, what are the reagent ions? The time dependence of fast-atom bombardment mass spectra and parallels to chemical ionization

The evaporation in vacuo of the matrices used and the particle-induced desorption of matrix molecules in fast-atom bombardment (FAB) contribute to a proposed high pressure region above the FAB matrix known as the selvedge region. If the neutral number density is sufficiently high, ions formed upon bombardment may undergo collisions with molecules, yielding matrix-related cluster ions and, in cases when the analyte is desorbed in neutral form, protonated and deprotonated analyte molecules. Similarities with the chemical ionization (CI experiment have been pointed out previously and are further developed here. If FAB is similar to CI, then the response depends on the structures of the reagent ions — those ions that react with gas phase analyte molecules. We consider here the time dependence of positive and negative ion FAB spectra to attempt to identify the reagent ions of FAB. A model is suggested for the FAB ion source which evaluates similarities to a CI source, as well as spatial aspects that are unique to desorption/ionization techniques.     

Study on the formation of an amoxicillin adduct with methanol using electrospray ion trap tandem mass spectrometry

The possibility of using the protonated methanol-adduct of antimicrobial amoxicillin for its identification and quantification at residue levels has been investigated, since it is impossible to completely suppress the formation of these adducts when methanol is present in the solvent system. This process has been monitored over time and as a function of concentration. It was determined that adducts were instantly formed and that the abundance of the protonated methanol-adduct at m/z 398 increased at the expense of the protonated molecule m/z 366 with storage time. The effect of several common solvents and mobile-phase additives on the ionization efficiency of amoxicillin and the formation of the methanol adduct has also been investigated. It was shown that the mass spectra of amoxicillin were strongly influenced by the solvent in which the analyte is dissolved and by the analyte concentration, as well as by the composition of mobile phase. Methanol was determined to be the best spray solvent, as it provided spectra with the lowest abundance of dimer ions. It was also determined that acetic acid as the mobile-phase additive provided the highest signal intensities, while ammonium acetate should not be used as an additive for the determination of amoxicillin at residue levels. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS), fragmentation of the protonated molecules and the protonated methanol-adduct ions, in both positive and negative ion mode, has been performed. The fragmentation was stable and strong product ion spectra were obtained. The linearity of the MS detector response, and that of the chromatographic method, was tested. Due to the linear behaviour it was concluded that the protonated methanol-adduct ion can be used for analytical purposes, i.e. for identification and quantification of amoxicillin at trace levels.     

On the mechanism of ion formation from the aqueous solutions irradiated with 3 microm IR laser pulses under atmospheric pressure

The mechanism of atmospheric pressure (AP) laser ionization of water and water/glycerol liquid samples at a 3-microm wavelength is studied experimentally. For the ion desorption, an in-house built Yb : YAG-pumped optical parametric oscillator (OPO) infrared (IR) laser has been coupled with AP MALDI ion source interfaced to an ion trap mass spectrometer (MS). It has been shown that water is primarily responsible for ion generation in water/glycerol samples, while glycerol increases the solution viscosity and decreases the water evaporation rate and sample losses. In contrast to AP UV-MALDI, the electric field in the case of AP IR-MALDI does not assist in ion production. It was found that the absence of the electrical field provides the optimum ionization condition both for water and water/glycerol liquid samples at the 3-microm laser irradiation. A two-stage ion formation mechanism, which includes the initial emission of microdroplets and release of molecular ions at the second stage, can explain the experimentally observed ion signal dependencies upon the voltage applied between MS inlet and the MALDI sample plate. Postionization using additional corona discharge APCI increases the observed signal by approximately 50%, which indicates that some portion of the analyte is desorbed in the form of neutral molecules.     

Novel method for [M + Cu]+ ion formation by matrix-assisted laser desorption ionization

Direct deposition of a MALDI sample onto a copper sample stage and irradiation with UV light (337 nm) produces copper adduct ions of both the matrix and analyte molecules. This technique for introducing Cu⁺ into the gas-phase avoids suppression of ion signal that accompanies addition of metal salts to the sample solution. We observe good correlation between the number of basic residues in peptides and the number of Cu⁺ ions that add to the peptide. For example, the peptide KRQHPG contains three basic residues and forms ions with up to three Cu⁺ adducts. Postsource decay experiments demonstrate that for arginine containing peptides, arginine anchors the Cu⁺ ion. That is, all metastable ions contain the arginine complexed to Cu⁺ and the only immonium ion observed is that of arginine–Cu⁺. In addition, preliminary calculations indicate that guanidine has the highest Cu⁺ ion affinity followed by histidine.     

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry

The mean initial velocities of analyte ions ranging in molecular weight from 1000 Da to 150 kDa and desorbed with a pulsed Er:YAG laser from various solid-state and liquid IR MALDI matrices were measured along with those of the matrix ions. Experiments with UV MALDI were performed for comparison in addition for a 2,5-dihydroxybenzoic acid preparation. Two different measurement principles were employed, (1) a delayed extraction method, relying on the initial velocity-dependent increase of flight times with delay time between laser and HV ion extraction pulse, and (2) a field-free drift method in which the first region of a two-stage ion source was varied in length and the flight times compared. The two methods yielded somewhat different values for the mean initial ion velocities. Based on a detailed discussion of the measurement principles it is suggested that the actual initial velocities of IR MALDI ions lie between the limits set by the two methods. The influences of the analyte-to-matrix ratio, laser fluence, and laser wavelength on the initial ion velocities were also investigated. Significant differences between the desorption mechanisms for liquid and solid-state matrices were observed.