Analysis of lipids with desorption atmospheric pressure photoionization‐mass spectrometry (DAPPI‐MS) and desorption electrospray ionization‐mass spectrometry (DESI‐MS)

In this article, the effect of spray solvent on the analysis of selected lipids including fatty acids, fat‐soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids has been studied by two different ambient mass spectrometry (MS) methods, desorption electrospray ionization‐MS (DESI‐MS) and desorption atmospheric pressure photoionization‐MS (DAPPI‐MS). The ionization of the lipids with DESI and DAPPI was strongly dependent on the spray solvent. In most cases, the lipids were detected as protonated or deprotonated molecules; however, other ions were also formed, such as adduct ions (in DESI), [M‐H]⁺ ions (in DESI and DAPPI), radical ions (in DAPPI), and abundant oxidation products (in DESI and DAPPI). DAPPI provided efficient desorption and ionization for neutral and less polar as well as for ionic lipids but caused extensive fragmentation for larger and more labile compounds because of a thermal desorption process. DESI was more suitable for the analysis of the large and labile lipids, but the ionization efficiency for less polar lipids was poor. Both methods were successfully applied to the direct analysis of lipids from pharmaceutical and food products. Although DESI and DAPPI provide efficient analysis of lipids, the multiple and largely unpredictable ionization reactions may set challenges for routine lipid analysis with these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Internal energy distributions in desorption electrospray ionization (DESI)

The internal energy distributions of typical ions generated by desorption electrospray ionization (DESI) were measured using the "survival yield" method, and compared with corresponding data for electrospray ionization (ESI) and electrosonic spray ionization (ESSI). The results show that the three ionization methods produce populations of ions having internal energy distributions of similar shapes and mean values (1.7-1.9 eV) suggesting similar phenomena, at least in the later stages of the process leading from solvated droplets to gas-phase ions. These data on energetics are consistent with the view that DESI involves "droplet pick-up" (liquid-liquid extraction) followed by ESI-like desolvation and gas-phase ion formation. The effects of various experimental parameters on the degree of fragmentation of p-methoxy-benzylpyridinium ions were compared between DESI and ESSI. The results show similar trends in the survival yields as a function of the nebulizing gas pressure, solvent flow rate, and distance from the sprayer tip to the MS inlet. These observations are consistent with the mechanism noted above and they also enable the user to exercise control over the energetics of the DESI ionization process, through manipulation of external and internal ion source parameters.     

Characterization of DESI-FTICR mass spectrometry - from ECD to accurate mass tissue analysis

Implementation of desorption electrospray ionization (DESI) technique on a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is described. Desorption electrospray technique is capable of the direct investigation of natural samples without any need for sample preparation or chromatographic separation. Since the DESI mass spectra of natural samples are very complex owing to the lack of preseparation or cleanup, the ideal mass spectrometric analyzer for these applications is a high-resolution instrument such as FTICR mass spectrometer. DESI was implemented by constructing an electronically controlled source framework comprising six linear moving stages and one rotating stage. A three-dimensional linear stage was used to accommodate samples, while another 3D linear stage equipped with rotating stage was used as a spray mount. A modified electrosonic sprayer was used as a primary electrospray device. DESI-FTICR setup was characterized with regard to geometrical, electrical and flow conditions using deposited peptide samples in range of 1-100 pmol gross deposited amount on glass and polymer surfaces. Optimized conditions enabled the routine acquisition of DESI-MS spectra on the instrument at 130 000 resolution in the broadband mode and with comparable sensitivity to data reported in the literature. Since the main significance of DESI-FTICR MS is the combination of intact tissue analysis, the capabilities of the technique were demonstrated by analyzing murine liver samples. Presence of lysophospholipids in the liver tissue was tentatively associated with the lipid metabolism taking place in liver. DESI-FTICR is also a promising technique in the field of peptide analysis due to capability of top-down sequencing using electron capture dissociation. As a proof-of-principle experiment, a small synthetic polypeptide containing 36 amino acids was ionized using DESI and was sequenced in the FTICR by means of ECD (electron capture dissociation) fragmentation. Spectra gave almost full sequence information in agreement with the known amino acid sequence of the species.     

Comparative studies of the behavior of neutral and ionic ferrocene derivatives under different conditions of electrospray ionization

We have studied the behavior of ferrocene CpFeCp (FcH), ferrocenium triiodide [FcH]⁺I₃⁻, dimethylaminomethylferrocene FcCH₂NMe₂ and its trimethylammonium salt [FcCH₂NMe₃]⁺I⁻ under the conventional conditions of electrospray ionization (ESI), when the substance solution is subjected to spraying, and in two versions of desorption electrospray ionization (DESI), when the sprayed solvent bombards the surface of solid or liquid samples. In addition to these techniques, the behavior of neutral compounds under conditions of electrospray ionization of vapors of the studied compounds in a gas phase (ESI_V) has been investigated. It has been shown using the examples of ferrocene and its dimethylaminomethyl derivative that the detection limits for these compounds occurring in a gas phase are comparable within an order of magnitude with their detection limits under the ESI and DESI conditions of solid and liquid samples. The high effectiveness of ionization of analyte vapors makes it possible to use the ESI method not only in combination with liquid (conventional ESI technology) and thin layer chromatography (DESI), but also with gas liquid chromatography (ESI_V). Thus, the electrospray ionization becomes a universal method allowing studies of a compound under the natural conditions in any state of aggregation, that is, solid, liquid, and gas. With the help of statistical methods for designing experiments (complete factorial experiment), quantitative evaluation of the influence of experimental parameters on the ion-formation processes under different ESI conditions has been carried out, which makes it possible to purposefully select the optimal conditions to record the ESI mass spectra with a minimum number of experiments. Moreover, analysis of the dependences of the mass spectra on the experimental parameters can serve as an instrument for studying the details of the ion-formation mechanisms depending upon different ways of ionization.     

Salt tolerance of desorption electrospray ionization (DESI)

The salt tolerance of desorption electrospray ionization (DESI) was systematically investigated by examining three different drug mixtures in the presence of 0, 0.2, 2, 5, 10, and 20% NaCl:KCl (1:1) from different surfaces. At physiological salt concentrations, the individual drugs in each mixture were observed in each experiment. Even at salt concentrations significantly above physiological levels, particular surfaces were effective in providing spectra that allowed the ready identification of the compounds of interest in low nanogram amounts. Salt adducts, which are observed even in the absence of added salt, could be eliminated by adding 0.1% 7 M ammonium acetate to the standard methanol:water (1:1) spray solvent. Comparison of the salt tolerance of DESI with that of electrospray ionization (ESI) demonstrated better signal/noise characteristics for DESI. The already high salt tolerance of DESI can be optimized further by appropriate choices of surface and spray solution.     

Enhanced detection of olefins using ambient ionization mass spectrometry: Ag<Superscript>+</Superscript> adducts of biologically relevant alkenes

Spray solvent doped with silver ions increases the ease of olefin detection by desorption electrospray ionization (DESI). Characteristic silver adducts were generated in up to 50 times greater abundance when compared to conventional DESI spray solvents for the biologically significant olefin, arachidonic acid, in the positive ion mode. In the analysis of 26 lipids, silver adduct formation was highly favorable for fatty acids, fatty acid esters and prostaglandins but not applicable to some other classes (e.g., polar lipids such as ceramide and its derivative cerebroside sulfate). An investigation exploring competitive Ag⁺ cationization with a mixture of components demonstrated that polyunsaturated compounds form Ag⁺ adducts most readily. Silver cationization allowed the distinction between three sets of isomers in the course of multiple-stage collision-induced dissociation, so providing insight into the location of the olefin bonds. A silver ion-doped solvent was used in DESI imaging of normal and tumor canine bladder tissue sections. The Ag⁺ fatty acid adducts permitted post facto differentiation between the normal and tumor regions. In addition, silver adduct formation in the course of DESI imaging of tissue sections revealed the presence of triacylglycerides, a class of compounds not previously identified through DESI imaging. A simple silver nitrate spray solvent has the potential to further improve DESI analysis of unsaturated biomolecules and other molecules containing π-bonds through selective silver cationization.     

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

Desorption electrospray ionization (DESI) mass spectrometry was evaluated for the characterization of glycerophospholipid standards, including glycerophosphocholine (GPCho), glycerophosphoglycerol (GPGro), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphoinositol (GPIns), cardiolipin (CL), and sphingolipid standards, including sulfatides (ST) and sphingomyelin (SM). Of specific interest were the effects of surface and solvent composition on signal stability and intensity, along with the ions observed in the full scan mode and the fragmentations seen upon collisional activation for each of the above classes. These experiments were performed without the addition of matrix compounds to the sample and were conducted in the free ambient environment at atmospheric pressure. The compounds GPSer, GPGro, GPIns, ST, and CL were best analyzed in the negative ion mode while PE was ionized efficiently in both positive and negative ion modes. SM and GPCho, which typically generate more abundant ions in the positive ion mode, could be analyzed in the negative ion mode by the addition of anionic reagents such as acetate to the spray solvent. Full scan DESI mass spectra and tandem (MS/MS) spectra for this representative set of physiological phospho/sphingolipids are presented. Similarities with other ionization methods in terms of fragmentation behavior were strong, although ambient ionization of untreated samples is only available with DESI. The effect of surface and solvent properties on signal intensity and stability were determined by depositing standard compounds on several different surfaces and analyzing with various proportions of methanol in the aqueous spray. Analysis was extended to complex mixtures of phospholipids and sphingolipids by examining the total lipid extract of porcine brain and by direct analysis of rat brain cryotome sections. These types of mixture analyses and molecular imaging studies are likely to represent major areas of application of DESI.     

Coupling of liquid chromatography with mass spectrometry by desorption electrospray ionization (DESI)

A liquid chromatography/mass spectrometry (LC/MS) method using desorption electrospray ionization (DESI) as a versatile interface has been established, which allows a wide range of elution flow rates, online derivatization via reactive DESI and further combination with electrochemistry.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography,using evaporative light-scattering and electrospray mass spectrometry for detection

A novel method was developed for the analysis of molecular species in neutral lipid classes, using separation by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 μm-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identification of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour.     

Direct detection of chlorpropham on potato skin using desorption electrospray ionization

Most pesticides, herbicides and other plant treatment agents are applied to the crop surface. Direct mass spectrometric methods, such as desorption electrospray ionization (DESI), offer new ways to analyze plant samples directly and rapidly. A strategy for the development and optimization of a DESI method for the direct determination of chemicals on complex surfaces is described. Chlorpropham (CP) was applied to potato surfaces as an example for a crop protection agent and analyzed using a self‐made DESI source. Aspects such as instrument selectivity, sensitivity and reproducibility were investigated. The MS⁴ fragmentation pattern of CP was analyzed to achieve the necessary detection selectivity, and is discussed in detail. Similar fragmentation was found in the ESI and DESI mass spectra, indicating that the mechanisms of ESI and DESI are closely related. A DESI method for semi‐quantification of CP on potatoes was developed. Detection limits of 6.5 µg/kg were found using MS/MS. The reproducibility, in the range of 12% (signal variation), appears to be sufficient for semi‐quantitative measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid analysis of free fatty acid composition in Brassica rapa L. and Brassica napus L. extracts by surface-assisted laser desorption/ionization time-of-flight mass spectrometry

A rapid and simple technique was developed to identify free fatty acids in mixtures by the surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) using powdered carbon aerogels as a “matrix”. A 3% solution of a neutral surfactant Triton X-100 was used as a fixing agent of carbon particles as well as a suppressor of background peaks. The extraction of free fatty acids from Brassica napus (rape) and Brassica rapa (turnip) seeds was performed by using both methanol-chloroform mixture and supercritical fluid extraction techniques. The quantitative analysis of the seed composition was carried out to demonstrate the effectiveness of the developed method. The detection limits were determined using a mixture of ten standard fatty acids and were found to be 3 ng of each acid per spot.     

The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry

The direct sampling feature of liquid sample desorption electrospray ionization (DESI) allows the ionization of liquid samples without adding acids/organic solvents (i.e., without sample pretreatment). As a result, it provides a new approach for probing protein conformation in solution. In this study, it has been observed that native protein ions are generated from proteins in water by DESI. Interestingly, the intensities of the resulting protein ions appear to be higher than those generated by ESI of the proteins in water or in ammonium acetate. For protein solutions that already contain acids/organic solvents, DESI can be used to investigate the influences of these denaturants on protein conformations and the obtained results are in good agreement with spectroscopic data. In addition, online monitoring of protein conformational changes by DESI is feasible; for instance, heat-induced unfolding of ubiquitin can be traced with DESI in water without influences of organic solvents/acids. This DESI method provides a new alternative tool for the study of protein conformation in solution.     

Effects of dopants in the imaging of mouse brain by desorption electrospray ionization/post-photoionization mass spectrometry

Desorption electrospray ionization/post-photoionization (DESI/PI) is a newly developed ionization method by the combination of DESI and post-photoionization for the simultaneous imaging of polar and nonpolar compounds in biological tissues. Dopants are of great importance in DESI/PI for the enhancement of signal intensities through ion–molecule reactions. In this work, to evaluate the performance of dopants in DESI/PI, an efficient homogenate model was developed, and four kinds of dopants (toluene, chlorobenzene, bromobenzene, and anisole) were tested using homogenate of mouse brain tissue as target sample. The influences of the dopants on the signal enhancements of different compounds were explained reasonably by the ionization mechanism. Then, the dopants with their optimum volume contents were applied to the mass spectrometry imaging (MSI). For a comprehensive imaging of various compounds with different polarities, methanol/toluene/formic acid (7:3:0.1) was chosen as the best choice. Finally, the stronger quantitative ability of DESI/PI with toluene as dopant for a few compounds in mouse brain tissue was demonstrated.     

Rapid in situ detection of alkaloids in plant tissue under ambient conditions using desorption electrospray ionization

Desorption electrospray ionization (DESI) mass spectrometry is applied to the in situ detection of alkaloids in the tissue of poison hemlock (Conium maculatum), jimsonweed (Datura stramonium) and deadly nightshade (Atropa belladonna). The experiment is carried out by electrospraying micro-droplets of solvent onto native or freshly-cut plant tissue surfaces. No sample preparation is required and the mass spectra are recorded under ambient conditions, in times of a few seconds. The impact of the sprayed droplets on the surface produces gaseous ions from organic compounds originally present in the plant tissue. The effects of operating parameters, including the electrospray high voltage, heated capillary temperature, the solvent infusion rate and the carrier gas pressure on analytical performance are evaluated and optimized. Different types of plant material are analyzed including seeds, stems, leaves, roots and flowers. All the previously reported alkaloids have been detected in C. maculatum, while fifteen out of nineteen known alkaloids for D. stramonium and the principal alkaloids of A. belladonna were also identified. All identifications were confirmed by tandem mass spectrometry. Results obtained show similar mass spectra, number of alkaloids, and signal intensities to those obtained when extraction and separation processes are performed prior to mass spectrometric analysis. Evidence is provided that DESI ionization occurs by both a gas-phase ionization process and by a droplet pick-up mechanism. Quantitative precision of DESI is compared with conventional electrospray ionization mass spectrometry (after sample workup) and the RSD values for the same set of 25 dicotyledonous C. maculatum seeds (one half of each seed analyzed by ESI and the other by DESI) are 9.8% and 5.2%, respectively.     

Transmission mode desorption electrospray ionization

A new mode of operation for desorption electrospray ionization (DESI) analysis of liquids or solid residues from evaporated solvents is presented. Unlike traditional DESI, the electrospray is not deflected off of a surface but instead is transmitted through a sampling mesh at a 0° angle between the electrospray tip, sample mesh, and capillary inlet of a mass spectrometer. In this configuration, deposited samples can be analyzed rapidly without rigorous optimization of spray distances or angles and without the preparation time associated with solvent evaporation. The new transmission mode desorption electrospray ionization (TM-DESI) technique is not applicable to bulk materials, but instead is a method designed to simplify the sample preparation process for liquid samples and sample extracts. The technique can reduce analysis time to seconds while consuming only microliters of sample. The results presented summarize the optimization of the technique, highlight key figures of merit for several model compounds, and illustrate potential applications to high throughput screening of liquid mixtures in both extraction solvents and biological matrices.     

Enhanced desorption ionization using oxidizing electrosprays

A signal enhancement of two orders of magnitude was achieved when reactive desorption electrospray ionization (DESI) was used to investigate copper(II) dibutyl dithiocarbamate, Cu(II)(bu2dtc)2, found in a specialized polymer. Cu(II) was oxidized to Cu(III) during the DESI experiment by oxidants in the spray solvent. Such oxidants could be present or formed during electrospray (e.g., O2) or deliberately added to the spray solvent (this approach is called reactive DESI). When a strong oxidizing agent (e.g., iodine) was added to the spray solvent, the signal increased by two orders of magnitude relative to the pure solvent spray. The correlation between the standard reduction potential of the oxidant and the signal intensity and signal to noise ratio of the product ion for various reagents, was tested and discussed. The observed DESI enhancements in rates of oxidation are not observed in homogeneous solution. The major peaks in the collision induced dissociation (CID) spectrum of the complex ion Cu(III)(bu2dtc)2]+ were identified using isotopic distributions and MS3 data.     

Reactive desorption electrospray ionization for selective detection of the hydrolysis products of phosphonate esters

Reactive desorption electrospray ionization (reactive DESI) is demonstrated to be a rapid and sensitive method for the direct detection of alkyl methylphosphonic acids, the hydrolysis products and metabolites of the chemical warfare (CW) agents VX (S-2-diisopropylaminoethyl-O-ethyl methylphosphonothiolate) and GB (sarin, isopropylmethyl phosphonofluoridate). Rapid and sensitive detection of these compounds is readily achieved by performing DESI from a solid surface; detection specificity is enhanced by implementation of a heterogeneous ion/molecule reaction using boric acid in the spray solvent. The reagent ion H(2)BO(3) (-) generated in the spray readily reacts with condensed-phase alkyl MPA to form anionic adducts. The specificity of this chemical reaction, together with the characteristic fragmentation patterns of the reaction products, supplies a highly discriminatory detection method for methylphosphonic acid (MPA), ethylphosphonic acid (EMPA) and isopropyl methylphosphonic acid (IMPA) in complex matrices.     

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.