Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.     

MALDI Matrices for the Analysis of Low Molecular Weight Compounds: Rational Design,Challenges and Perspectives

The analysis of low molecular weight (LMW) compounds is of great interest to detect small pharmaceutical drugs rapidly and sensitively, or to trace and understand metabolic pathways. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) plays a central role in the analysis of high molecular weight (bio)molecules. However, its application for LMW compounds is restricted by spectral interferences in the low m/z region, which are produced by conventional organic matrices. Several strategies regarding sample preparation have been investigated to overcome this problem. A different rationale is centred on developing new matrices which not only meet the fundamental requirements of good absorption and high ionization efficiency, but are also vacuum stable and “MALDI silent”, i. e., do not give matrix-related signals in the LMW area. This review gives an overview on the rational design strategies used to develop matrix systems for the analysis of LMW compounds, focusing on (i) the modification of well-known matrices, (ii) the search for high molecular weight matrices, (iii) the development of binary, hybrid and nanomaterial-based matrices, (iv) the advance of reactive matrices and (v) the progress made regarding matrices for negative or dual polarity mode.     

Quantitation Of The Local Anesthetic Dibucaine With Gas Chromatography / Mass Spectrometry

Abstract

A chemical ionization and an electron impact GC/MS assaying approach is presented for determining dibucaine concentrations in biological fluids. Both use deuterium-labeled drug as the internal standard and rely on the same sample extraction and sample preparation procedure. Under chemical ionization conditions (CH₄), the assaying limits are in the range of 1–80 ng/ml of serum. Under electron impact conditions, the analytical range is 20–800 ng/ml. The chemical ionization procedure has been found suitable for monitoring drug levels in man. One volunteer, who received a single 5-mg oral dose, showed peak serum drug concentrations of 23 ng/ml attained 2 hr after drug administration. The biologic half-life (ß phase) was 11 hr.     

Analysis of various organic and organometallic compounds using nanostructure-assisted laser desorption/ionization time-of-flight mass spectrometry (NALDI-TOFMS)

Nanostructure-assisted laser desorption/ionization time-of-flight mass spectrometry (NALDI-TOFMS) has been developed recently as a matrix-free/surface-assisted alternative to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The NALDI surface of silicon nanowires is already very effective for the analysis of small to medium sized, polar organic molecules in positive ion mode. The current study examined this technology for the analysis of several nonpolar organic, organometallic, and ionic compounds in positive ion mode, as well as a fluorinated compound and various acids in negative ion mode. NALDI data are compared and contrasted with MALDI data for the same compounds, and the higher sensitivity of NALDI is highlighted by the successful characterization of two porphyrins for a sample amount of 10 amol per spot.     

Coffee-ring effects in laser desorption/ionization mass spectrometry

This report focuses on the heterogeneous distribution of small molecules (e.g. metabolites) within dry deposits of suspensions and solutions of inorganic and organic compounds with implications for chemical analysis of small molecules by laser desorption/ionization (LDI) mass spectrometry (MS). Taking advantage of the imaging capabilities of a modern mass spectrometer, we have investigated the occurrence of “coffee rings” in matrix-assisted laser desorption/ionization (MALDI) and surface-assisted laser desorption/ionization (SALDI) sample spots. It is seen that the “coffee-ring effect” in MALDI/SALDI samples can be both beneficial and disadvantageous. For example, formation of the coffee rings gives rise to heterogeneous distribution of analytes and matrices, thus compromising analytical performance and reproducibility of the mass spectrometric analysis. On the other hand, the coffee-ring effect can also be advantageous because it enables partial separation of analytes from some of the interfering molecules present in the sample. We report a “hidden coffee-ring effect” where under certain conditions the sample/matrix deposit appears relatively homogeneous when inspected by optical microscopy. Even in such cases, hidden coffee rings can still be found by implementing the MALDI-MS imaging technique. We have also found that to some extent, the coffee-ring effect can be suppressed during SALDI sample preparation.     

Continuous on-line determination of methyl tert-butyl ether in water samples using ion mobility spectrometry

A rapid analytical procedure for the on-line determination of methyl tert-butyl ether (MTBE) in water samples was developed. A new membrane extraction unit was used to extract the MTBE from water samples. The concentration of MTBE was determined using ion mobility spectrometry with 63Ni ionization and corona discharge ionization without chromatographic separation. Both ionization methods permit the sensitive determination of MTBE. A detection limit of 100 microg/L was established for the on-line procedure. Neither the inorganic compounds, humic substances nor gasoline were found to exert a significant influence on the peak intensity of the MTBE. The screening procedure can be used for concentrations of monoaromatic compounds (benzene, toluene, xylene) up to 600 microg/L. No sample preparation is required and the analysis results are available within 5 min. In order to determine concentrations between 10 microg/L and 100 microg/L, a discontinuous procedure was developed on the basis of the same experimental set-up.     

Evaluation of multidimensional capillary electrophoretic methodologies for determination of amino bisphosphonate pharmaceuticals

Alendronate and pamidronate are amino bisphosphonate analogues of pyrophosphate used for the treatment of a variety of bone diseases. Analysis of these compounds is problematic due to their polar ionic nature, lack of a suitable chromophore and chelation properties and current analytical approaches involve extensive sample preparation and derivatization procedures. The potential of multidimensional capillary electrophoretic methodological approaches, which eliminate sample preparation have been evaluated for the analysis of these compounds both in aqueous and urinary matrices. Capillary isotachophoresis (cITP) was employed as a pre-separation and on-line sample concentration step prior to analytical determination using either cITP or capillary zone electrophoresis (CZE) with conductivity detection. Single cITP, cITP-cITP and cITP-CZE approaches were partially validated with respect to repeatability, recovery and linearity of response for both compounds. The increases in sensitivity achievable through increasing injection volume from 30 to 300muL may render such strategies appropriate for determination of these agents at biologically relevant concentrations with minimal sample work-up.     

LC-MS in analytical toxicology: some practical considerations

Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the influence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology.     

Combining desorption electrospray ionization mass spectrometry and nuclear magnetic resonance for differential metabolomics without sample preparation

Desorption electrospray ionization mass spectrometry (DESI-MS) and nuclear magnetic resonance (NMR) spectroscopy are used to provide data on urine examined without sample preparation to allow differentiation between diseased (lung cancer) and healthy mice. Principal component analysis (PCA) is used to shortlist compounds with potential for biomarker screening which are responsible for significant differences between control urine samples and samples from diseased animals. Similar PCA score plots have been achieved by DESI-MS and NMR, using a subset of common detected metabolites. The common compounds detected by DESI and NMR have the same changes in sign of their concentrations thereby indicating the usefulness of corroborative analytical methods. The effects of different solvents and surfaces on the DESI mass spectra are also evaluated and optimized. Over 80 different metabolites were successfully identified by DESI-MS and tandem mass spectrometry experiments, with no prior sample preparation.     

A comparison of EDI with solvent-free MALDI and LDI for the analysis of organic pigments

To evaluate the applicability of EDI to material analysis as a new ionization method, a comparison of EDI with solvent-free matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) was made for the analysis of organic pigments, e.g. Pigment Yellow 93, Pigment Yellow 180, and Pigment Green 36, as test samples, which are poorly soluble in standard solvents. In EDI, the samples were prepared in two ways: deposition of suspended samples in appropriate solvents and dried on the substrate, and the direct deposition of the powder samples on the substrate. No matrices were used. Both sample preparation methods gave similar mass spectra. Equally strong signals of [M + H](+) and [M - H](-) ions were observed with some fragment ions for azo pigments in the respective positive or negative mode of operation. For the powder sample of the phthalocyanine pigment PG36, M(+*) and [M + H](+) in the positive mode and M(-*) in the negative mode of operation were observed as major ions. Positive-mode, solvent-free MALDI gave M(+), [M + H](+) and [M + Na](+) and negative mode gave [M - H](-) depending on the sample preparation. As solvent-free MALDI, EDI was also found to be an easy-to-operate, versatile method for the samples as received.     

Novel approaches in analysis of Fusarium mycotoxins in cereals employing ultra performance liquid chromatography coupled with high resolution mass spectrometry

Rapid, simple and cost-effective analytical methods with performance characteristics matching regulatory requirements are needed for effective control of occurrence of Fusarium toxins in cereals and cereal-based products to which they might be transferred during processing. Within this study, two alternative approaches enabling retrospective data analysis and identification of unknown signals in sample extracts have been implemented and validated for determination of 11 major Fusarium toxins. In both cases, ultra-high performance liquid chromatography (U-HPLC) coupled with high resolution mass spectrometry (HR MS) was employed. ¹³C isotopically labeled surrogates as well as matrix-matched standards were employed for quantification. As far as time of flight mass analyzer (TOF-MS) was a detection tool, the use of modified QuEChERS (quick easy cheap effective rugged and safe) sample preparation procedure, widely employed in multi-pesticides residue analysis, was shown as an optimal approach to obtain low detection limits. The second challenging alternative, enabling direct analysis of crude extract, was the use of mass analyzer based on Orbitrap technology. In addition to demonstration of full compliance of the new methods with Commission Regulation (EC) No. 401/2006, also their potential to be used for confirmatory purposes according to Commission Decision 2002/657/EC has been critically assessed.     

Gas chromatography coupled to high-resolution time-of-flight mass spectrometry to analyze trace-level organic compounds in the environment, food safety and toxicology

Gas chromatography coupled to high-resolution time-of flight mass spectrometry (GC-HR-TOF-MS) is a powerful analytical technique with excellent capabilities due to its high sensitivity in full-spectrum-acquisition mode together with its resolving power and accurate-mass measurements. These features make this technique very attractive in qualitative analysis, especially for wide-scope screening of a large number of organic contaminants and residues at trace levels.Full-spectrum MS allows data processing, in principle, of an unlimited number of compounds in samples, as no analyte-specific information is required before the injection. Also, as all data remain available, retrospective analysis is always possible without the need to reinject the sample - an important advantage of full-spectrum MS techniques. Despite these advantages, GC-HR-TOF-MS has rarely been applied, so we expect promising results in different applied fields in the years ahead.We discuss in detail the characteristics and the potential of GC-HR-TOF-MS. We describe different analytical strategies from wide-scope target screening to investigation of unknowns in biology, the environment and food safety. Recent instrumental developments (e.g., high-speed analog-to-digital converter or soft ionization sources) and advances in software for processing the huge amount of data available open up new prospects, making GC-TOF-MS one of the most promising techniques to investigate the presence of organic compounds in different fields.     

Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20 kDa) proteins

Three sample preparation strategies commonly employed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS) of whole bacterial cells were investigated for the detection of high mass signals; these included the dried droplet, the seed-layer/two-layer, and the bottom-layer methods. Different sample preparation approaches favoured the detection of high- or low-mass proteins. The low-mass peaks were best detected using the bottom-layer method. By contrast, the dried droplet method using a solvent with higher water content, and hence effecting a slower crystallization process, gave the best results for the detection of high-mass signals. Signals up to m/z 158 000 could be detected with this methodology for Bacillus sphaericus. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the same extracts used for MALDI-TOFMS showed bands in the molecular weight range in which high-mass peaks were observed in MALDI-MS, suggesting that the high-mass signals are not polymeric adducts of low-mass protein monomers. In addition, one of the high molecular weight proteins (approximately 126 kDa) was putatively identified as an S-layer protein by an in-gel tryptic digest. The bacterial samples spotted on the target wells for MALDI-TOFMS, using the different sample preparation strategies, were examined under a scanning electron microscope and differences were observed between the different strategies, suggesting that the nature of the crystals and the distribution of the analytes amidst the crystals could influence the spectral pattern observed in MALDI-TOFMS of whole bacterial cells. Finally, evidence is presented to indicate that, although the determinants are intact cells, cell lysis occurs both before and during the MALDI process.     

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.

Analysis of complex mixtures by photoionization mass spectrometry with a vacuum-ultraviolet hydrogen-laser source

Trace organic analysis of a complex matrix presents one of the most challenging problems in analytical mass spectrometry. Inselective electron-impact ionization often requires extensive sample clean-up to isolate the analyte from the matrix. Sample preparation can be greatly reduced when a hydrogen laser is used for selective photoionization of only a small fraction of the compounds introduced into the ion source. This device produces parent ions only for all compounds with ionization potentials that lie below a threshold value limited by the photon energy of 7.8 eV. The only observed interference arises from electron-impact ionization when scattered laser radiation interacts with metal surfaces, producing electrons which are then accelerated by potential fields inside the source. These can be suppressed to levels acceptable for practical analysis through proper instrumental design. Results are presented which indicate the ability of this ion source to discriminate against interfering matrix components in simple extracts from real samples such as brewed coffee, beer, and urine.     

The use of nonpolar matrices for matrix-assisted laser desorption/ionization mass spectrometric analysis of high boiling crude oil fractions

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with nonpolar matrices has been investigated for its applicability to the characterization of atmospheric resid crude oil fractions. The data obtained by use of nonpolar matrices was compared with that from polar matrices as well as from direct LDI-MS and field ionization mass spectrometry. Nonpolar matrices, such as anthracene or 9-cyanoanthracene, yield only a single radical molecular cation upon LDI. Thus, no interfering matrix-related ions are present during the MALDI-TOFMS analysis of the crude oil sample. Nonpolar matrices yield molecular mass distributions from linear mode MALDI-TOFMS that are comparable to distributions found with LDI-MS. An advantage of nonpolar matrices is the increased production of analyte ions, which allows reflectron mode MALDI-TOFMS to be performed. Nonpolar matrices are also shown to be less sensitive to solvent and sample preparation conditions than conventional polar matrices. These results suggest that nonpolar matrices may be favorable alternatives to more traditional polar or acidic matrices commonly used in the MALDI mass spectral characterization of crude oil related samples.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of Hanford-Related Organics Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low-molecular phosphate compounds found in Hanford tank wastes. The mass spectra of these compounds indicate protonated peaks as well as sodium adducts. Analytical methods presently utilized for the analysis of the phosphate-related organics are both time consuming and labor intensive. A promising alternative is MALDI/TOFMS. The MALDI process produces both positive and negative ions directly and very little sample is required. In addition, there is limited sample preparation and minimal hazardous waste production.     

Analysis of multiple pesticide residues in tobacco using pressurized liquid extraction, automated solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry

A new method was developed for the analysis of pesticide residues in tobacco. The objective was to significantly increase the number of samples that can be processed by the laboratory and to enable the extension of the current coverage to additional pesticides. A new analytical approach was therefore defined based on two main axes, the automation of the sample preparation and the selectivity of the analyte detection using tandem mass spectrometry. This latter aspect reduces the stringency of the requirements placed on the clean-up of the extracts and on the chromatographic resolution when less selective detectors are used. The extraction of the analytes from the matrix is performed using the pressurized liquid extraction technique. Tobacco samples are extracted at elevated temperature and pressure (100 C and 100 atm; 1 atm = 101,325 Pa) using acetone as an extraction solvent. The resulting extract is then concentrated using a Vortex evaporator. Three different solid-phase extraction (SPE) procedures, adjusted to the chemical properties of the different active ingredients to be measured, are applied to the concentrated extract, thus leading to three extract fractions. The first fraction contains such main classes of active ingredients as organohalogenated and 2,6-dinitroaniline compounds while the second one collects the organophosphorus and acylalanines residues; these two fractions are analyzed by capillary gas chromatography coupled to tandem mass spectrometry using negative chemical ionization and electron impact ionization in the positive mode, respectively. The third extract fraction gathers the N-methylcarbamates residues which are analyzed by HPLC with post-column derivatization and fluorescence detection. The different sample preparation stages from extraction to SPE clean-up have been automated through the use of recent analytical technologies. In combination with the analysis by tandem mass spectrometry, this provided a potential for a high sample throughput.     

Development of a Liquid Chromatography Tandem Mass Spectrometry Method for Identification of Flavonoids in Ginkgo biloba

A rapid, selective and sensitive method for analysis of trace flavonoids and its glycoside derivatives in ginkgo has proposed. Ultrasonic‐assisted extraction of sample preparation was adopted to extract trace flavonoids in ginkgo leaf and its processed product. The compounds were identified using liquid chromatography negative electrospray ionization triple quadrupole tandem mass spectrometry (MS/MS). The neutral loss scan mode of MS/MS was used to screen flavonoid compounds and those compounds with acid group, or having rhamnosyl, glucosyl, or coumaroyl moiety in the samples. The successive data‐dependent product ion scan mode of MS/MS was used to identify the structure of the components. The analytical results represented three aglycone flavonoids and seven flavonoid glycosides in ginkgo. The method detection limits were evaluated for the analytes analyzed in the range of 0.88 to 2.67 μg/mL.