Screening of Complicated Matrixes with Paper Assisted Ultrasonic Spray Ionization Mass Spectrometry

To analyze compounds in complicated matrixes using mass spectrometry, we describe a novel ambient ionization approach, termed paper assisted ultrasonic spray ionization (PAUSI). The ionization process is based on the ultrasonic vibration of the piezoelectric ceramic disk, on which the samples are placed. Porous materials are utilized to generate fine initial droplet, which could alleviate matrix effect during ionization process for complicated matrix. PAUSI was evaluated as an attractive tool to screen analytes from complicated matrixes, such as (1) bovine serum with NaCl 150 g/L, (2) viscous samples, and (3) biological fluid, without any sample preparation. Moreover, it provides great advantage in simplifying the mass spectrometry analysis process, and the ionization device is inexpensive and easy to operate.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Imaging Source Coupled to a FT-ICR Mass Spectrometer

Mass spectrometry imaging (MSI) allows for the direct monitoring of the abundance and spatial distribution of chemical compounds over the surface of a tissue sample. This technology has opened the field of mass spectrometry to numerous innovative applications over the past 15 years. First used with SIMS and MALDI MS that operate under vacuum, interest has grown for mass spectrometry ionization sources that allow for effective imaging but where the analysis can be performed at ambient pressure with minimal or no sample preparation. We introduce here a versatile source for MALDESI imaging analysis coupled to a hybrid LTQ-FT-ICR mass spectrometer. The imaging source offers single shot or multi-shot capability per pixel with full control over the laser repetition rate and mass spectrometer scanning cycle. Scanning rates can be as fast as 1 pixel/second and a spatial resolution of 45 μm was achieved with oversampling.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

CO2 laser ionization of acoustically levitated droplets

For many analytical purposes, direct laser ionization of liquids is desirable. Several studies on supported droplets, free liquid jets, and ballistically dispensed microdroplets have been conducted, yet detailed knowledge of the underlying mechanistics in ion formation is still missing. This contribution introduces a simple combination of IR-MALDI mass spectrometry and an acoustical levitation device for contactless confinement of the liquid sample. The homebuilt ultrasonic levitator supports droplets of several millimeters in diameter. These droplets are vaporized by a carbon dioxide laser in the vicinity of the atmospheric pressure interface of a time of flight mass spectrometer. The evaporation process is studied by high repetition rate shadowgraphy experiments elucidating the ballistic evaporation of the sample and revealing strong confinement of the vapor by the ultrasonic field of the trap. Finally, typical mass spectra for pure glycerol/water matrix and lysine as an analyte are presented with and without the addition of trifluoracetic acid, and the ionization mechanism is briefly discussed. The technique is a promising candidate for a reproducible mass spectrometric detection scheme for the field of microfluidics.

Analytical Properties of Solid-substrate Electrospray Ionization Mass Spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) uses a capillary for sample loading and ionization. Along with the development of ambient ionization techniques, ESI-MS using noncapillary emitters has attracted more interest in recent years. Following our recent report on ESI-MS using wooden tips (Anal. Chem. 83, 8201–8207 (2011)), the technique was further investigated and extended in this study. Our results revealed that the wooden tips could serve as a chromatographic column for separation of sample components. Sequential and exhaustive ionization was observed for proteins and salts on wooden tips with salts ionized sooner and proteins later. Nonconductive materials that contain microchannels/pores could be used as tips for ESI-MS analysis with sample solutions loaded to the sharp-ends only, since rapid diffusion of sample solutions by capillary action would enable the tips to become conductive. Tips of inert materials such as bamboo, fabrics, and sponge could be used for sample loading and ionization, while samples such as tissue, mushroom, and bone could form tips to induce ionization for direct analysis with application of a high voltage.

Gas chromatography/multiphoton ionization/time-of-flight mass spectrometry using a femtosecond laser

A laser can be used for the selective excitation and subsequent ionization of a molecule with an absorption band at the laser wavelength. This technique of multiphoton ionization (MPI), when combined with time-of-flight mass spectrometry (TOF-MS), permits the efficient detection of induced ions in mass analysis. This combination of MPI/TOF-MS can be coupled with gas chromatography (GC) to achieve even more enhanced selectivity. Thus, GC/MPI/TOF-MS can be employed for trace analysis of samples containing numerous chemical species. A variety of laser sources have been used for this purpose. Since molecules that are classified as environmental pollutants frequently contain chlorine and bromine atoms, the lifetime of the excited state can be decreased by energy transfer from the singlet to triplet levels by spin–orbit interaction. A high-power femtosecond laser with a pulse width shorter than the lifetime of the analyte molecule provides femtogram or even subfemtogram detection limits, which have not yet been achieved using the most sensitive high-resolution double-focus sector-type mass spectrometers. Numerous environmental pollutants such as dioxins in soils and pesticides in foods have been successfully quantified using GC/MPI/TOF-MS, and this technique has proven itself to be a useful and practical method for trace analysis.

Multiclass method for antimicrobial analysis in animal feeds by liquid chromatography–tandem mass spectrometry

A rapid multiclass method that covers 50 antimicrobials from 13 different families in animal feeds was developed. Samples were extracted using a mixture of methanol, acetonitrile and a McIlvaine buffer combined with sonication. Feed extracts were simply diluted prior to injection, since the clean-up strategies that were tested, based on either solid-phase extraction or dispersive solid-phase extraction, were ineffective at minimizing matrix-related signal suppression/enhancement. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry using an electrospray ionization source operating in positive and negative modes. For the quantification, matrix-fortified standard calibration curves were used to compensate for matrix effects and losses in sample preparation. The method was validated in-house in pig, poultry and cattle feed matrices and showed satisfactory performance characteristics. Thus, the proposed approach was suitable for application in a routine high-throughput laboratory for the official control of feeds.

Minimization of Fragmentation and Aggregation by Laser Desorption Laser Ionization Mass Spectrometry

Measuring average quantities in complex mixtures can be challenging for mass spectrometry, as it requires ionization and detection with nearly equivalent cross-section for all components, minimal matrix effect, and suppressed signal from fragments and aggregates. Fragments and aggregates are particularly troublesome for complex mixtures, where they can be incorrectly assigned as parent ions. Here we study fragmentation and aggregation in six aromatic model compounds as well as petroleum asphaltenes (a naturally occurring complex mixture) using two laser-based ionization techniques: surface assisted laser desorption ionization (SALDI), in which a single laser desorbs and ionizes solid analytes; and laser ionization laser desorption mass spectrometry (L²MS), in which desorption and ionization are separated spatially and temporally with independent lasers. Model compounds studied include molecules commonly used as matrices in single laser ionization techniques such as matrix assisted laser desorption ionization (MALDI). We find significant fragmentation and aggregation in SALDI, such that individual fragment and aggregate peaks are typically more intense than the parent peak. These fragment and aggregate peaks are expected in MALDI experiments employing these compounds as matrices. On the other hand, we observe no aggregation and only minimal fragmentation in L²MS. These results highlight some advantages of L²MS for analysis of complex mixtures such as asphaltenes.

Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry

Cell transfer by contact printing coupled with carbon-substrate-assisted laser desorption/ionization was used to directly profile and image secondary metabolites in trichomes on leaves of the wild tomato Solanum habrochaites. Major specialized metabolites, including acyl sugars, alkaloids, flavonoids, and terpenoid acids, were successfully detected in positive ion mode or negative ion mode, and in some cases in both modes. This simple solvent-free and matrix-free sample preparation for mass spectrometry imaging avoids tedious sample preparation steps, and high-spatial-resolution images were obtained. Metabolite profiles were generated for individual glandular trichomes from a single Solanum habrochaites leaf at a spatial resolution of around 50 μm. Relative quantitative data from imaging experiments were validated by independent liquid chromatography–mass spectrometry analysis of subsamples from fresh plant material. The spatially resolved metabolite profiles of individual glands provided new information about the complexity of biosynthesis of specialized metabolites at the cellular-resolution scale. In addition, this technique offers a scheme capable of high-throughput profiling of metabolites in trichomes and irregularly shaped tissues and spatially discontinuous cells of a given cell type.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

A New Matrix Assisted Ionization Method for the Analysis of Volatile and Nonvolatile Compounds by Atmospheric Probe Mass Spectrometry

Matrix assisted ionization of nonvolatile compounds is shown not to be limited to vacuum conditions and does not require a laser. Simply placing a solution of analyte dissolved with a suitable matrix such as 3-nitrobenzonitrile (3-NBN) or 2,5-dihydroxyacetophenone on a melting point tube and gently heating the dried sample near the ion entrance aperture of a mass spectrometer using a flow of gas produces abundant ions of peptides, small proteins, drugs, and polar lipids. Fundamental studies point to matrix-mediated ionization occurring prior to the entrance aperture of the mass spectrometer. The method is analytically useful, producing peptide mass fingerprints of bovine serum albumin tryptic digest consuming sub-picomoles of sample. Application of 100 fmol of angiotensin I in 3-NBN matrix produces the doubly and triply protonated molecular ions as the most abundant peaks in the mass spectrum. No carryover is observed for samples containing up to 100 pmol of this peptide. A commercial atmospheric samples analysis probe provides a simple method for sample introduction to an atmospheric pressure ion source for analysis of volatile and nonvolatile compounds without using the corona discharge but using sample preparation similar to matrix-assisted laser desorption/ionization.

High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue

An atmospheric pressure laser desorption/ionization mass spectrometry imaging ion source has been developed that combines high spatial resolution and high mass resolution for the in situ analysis of biological tissue. The system is based on an infrared laser system working at 2.94 to 3.10 μm wavelength, employing a Nd:YAG laser-pumped optical parametrical oscillator. A Raman-shifted Nd:YAG laser system was also tested as an alternative irradiation source. A dedicated optical setup was used to focus the laser beam, coaxially with the ion optical axis and normal to the sample surface, to a spot size of 30 μm in diameter. No additional matrix was needed for laser desorption/ionization. A cooling stage was developed to reduce evaporation of physiological cell water. Ions were formed under atmospheric pressure and transferred by an extended heated capillary into the atmospheric pressure inlet of an orbital trapping mass spectrometer. Various phospholipid compounds were detected, identified, and imaged at a pixel resolution of up to 25 μm from mouse brain tissue sections. Mass accuracies of better than 2 ppm and a mass resolution of 30,000 at m/z?=?400 were achieved for these measurements.

Detection of Amine Impurity and Quality Assessment of the MALDI Matrix α-Cyano-4-Hydroxy-Cinnamic Acid for Peptide Analysis in the amol Range

We have studied sample preparation conditions to increase the reproducibility of positive UV-MALDI-TOF mass spectrometry of peptides in the amol range. By evaluating several α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix batches and preparation protocols, it became apparent that two factors have a large influence on the reproducibility and the quality of the generated peptide mass spectra: (1) the selection of the CHCA matrix, which allows the most sensitive measurements and an easier finding of the “sweet spots,” and (2) the amount of the sample volume deposited onto the thin crystalline matrix layer. We have studied in detail the influence of a contaminant, coming from commercial CHCA matrix batches, on sensitivity of generated peptide mass spectra in the amol as well as fmol range of a tryptic peptide mixture. The structure of the contaminant, N,N-dimethylbutyl amine, was determined by applying MALDI-FT-ICR mass spectrometry experiments for elemental composition and MALDI high energy CID experiments utilizing a tandem mass spectrometer (TOF/RTOF). A recrystallization of heavily contaminated CHCA batches that reduces or eliminates the determined impurity is described. Furthermore, a fast and reliable method for the assessment of CHCA matrix batches prior to tryptic peptide MALDI mass spectrometric analyses is presented.

Rapid and sensitive HILIC–MS/MS analysis of carnitine and acetylcarnitine in biological fluids

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography–tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

Distribution study of atorvastatin and its metabolites in rat tissues using combined information from UHPLC/MS and MALDI-Orbitrap-MS imaging

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α?=?0.05) affect the recoveries of ergot alkaloids.

Resonance-enhanced multiphoton ionization with circularly polarized light: chiral carbonyls

An introduction to the principle and possibilities of the new method of circular dichroism laser mass spectrometry is given and its state of development is reviewed. This method allows enantiosensitive, mass-selective probing of chiral molecules. It is based on the combination of resonance-enhanced multiphoton ionization with circularly polarized light and specially modified time-of-flight mass spectrometry. As an example, application to carbonyls is presented.

Toward high spatial resolution sampling and characterization of biological tissue surfaces using mass spectrometry

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.

Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe

A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry.

Flow injection of liquid samples to a mass spectrometer with ionization under vacuum conditions: a combined ion source for single-photon and electron impact ionization

Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI) are the most important techniques for the ionization of liquid samples. However, working under atmospheric pressure conditions, all these techniques involve some chemical rather than purely physical processes, and therefore, side reactions often yield to matrix-dependent ionization efficiencies. Here, a system is presented that combines both soft single-photon ionization (SPI) and hard 70 eV electron impact ionization (EI) of dissolved compounds under vacuum conditions. A quadrupole mass spectrometer was modified to enable direct EI, a technique developed by Cappiello et al. to obtain library-searchable EI mass spectra as well as soft SPI mass spectra of sample solutions. An electron beam-pumped rare gas excimer lamp working at 126 nm was used as well as a focusable vacuum UV light source for single-photon ionization. Both techniques, EI and SPI, were applied successfully for flow injection experiments providing library-matchable EI fragment mass spectra and soft SPI mass spectra, showing dominant signals for the molecular ion. Four model compounds were analyzed: hexadecane, propofol, chlorpropham, and eugenol, with detection limits in the picomolar range. This novel combination of EI and SPI promises great analytical benefits, thanks to the possibility of combining database alignment for EI data and molecular mass information provided by SPI. Possible applications for the presented ionization technology system are a matrix-effect-free detection and a rapid screening of different complex mixtures without time-consuming sample preparation or separation techniques (e.g., for analysis of reaction solutions in combinatorial chemistry) or a switchable hard (EI) and soft (SPI) MS method as detection step for liquid chromatography.