Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.     

Determination of plasma bile acids by capillary gas-liquid chromatography-electron capture negative chemical ionization mass fragmentography

Combined capillary gas-liquid chromatography-electron capture negative chemical ionization mass spectrometry of pentafluorobenzyl ester-TMSi ether derivatives of bile acids and isotope dilution using deuterated internal standards are introduced as a sensitive and selective analysis technique for plasma bile acids. As a result of the high ionization efficiency of pentafluorobenzyl derivatives under electron capturing conditions and minimal fragmentation, the detection limit of this technique is low: 1 pg for each bile acid. The high sensitivity enabled the detection and quantitation of atypical bile acids in 200-microliters aliquots of plasma from fasting healthy adults as exemplified by trihydroxycoprostanic acid (0.002 +/- 0.001 mumol/l) and dihydroxycoprostanic acid (0.013 +/- 0.002 mumol/l).     

Quantitative field desorption mass spectrometry. V. Discussion of methodology and examples of applications

The possibilities for the application of field desorption mass spectrometry in quantitative analyses are described and evaluated. The advantages of and the sources of errors in the use of different standards as well as in the application of different methods such as photographic detection, single ion monitoring, repetitive scanning, selected ion monitoring, and double ion detection are illustrated by representative examples. Sensitivity and precision of the different techniques are evaluated. Most importantly, the use of stable isotope labelled compounds as internal standards has enabled quantitative determination with good precision, accuracy, and sensitivity. In order to demonstrate the capabilities of the methods, examples of applications are presented and the scope of quantitative analysis with field desorption mass spectrometry is discussed.     

Urine profile analysis by field desorption mass spectrometry, a technique for detecting metabolites of xenobiotics. Application to 3,5-dinitro-2-hydroxytoluene

A technique for the detection of biotransformation products of xenobiotics in crude urine extracts by field desorption mass spectrometric profile analysis is described. The method comprises determination of peak profiles of a series of blank and test samples using low resolution field desorption mass spectrometry, comparison of averaged peak profiles and noise reduction by means of Fisher and ratio weighting of peak intensities. Application of the technique to 3,5-dinitro-2-hydroxytoluene has resulted in the detection of two hitherto unknown metabolites in rat urine. By thin-layer co-chromatography, high resolution electron impact mass spectrometry and thin-layer chromatographic/field desorption mass spectrometric analysis they could be identified as 3,5-dinitro-2-hydroxybenzenemethanol and 3,5-diacetamido-2-hydroxytoluene.     

Accurate determination of the molecular weight of the major surface layer protein isolated from Clostridium thermosaccharolyticum by time-of-flight mass spectrometry.

Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da. The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2%. This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.     

基体辅助激光解吸电离质谱法及其应用

基体辅助激光解吸电离质谱是近几年才发展起来的一种新技术,它在生命科学研究中具有广阔的应用前景．对基体辅助激光解吸电离质谱技术的基本原理,运用基体辅助激光解吸电离质谱法研究蛋白质分子量的测定,蛋白质混合物的分离鉴定以及用基体辅助激光解吸电离质谱技术进行超快速的蛋白质序列分析和DNA序列分析的可行性和存在的问题作了介绍和讨论．     

RNAA as compared to ICP-MS for the analysis of normal human serum

The merits of radiochemical neutron activation analysis (RNAA) and inductively coupled plasma mass spectrometry (ICP-MS) are critically discussed for the determination of trace and ultratrace elements in normal human serum. For RNAA, two semiautomated procedures, allowing the determination of up to 18 elements, are briefly described. ICP-MS has a series of interesting features for the determination of trace elements. Matrix and spectral interferences can mostly be avoided or corrected for. After a simple 5- or 10-fold dilution and addition of an internal standard, more than 20 elements can be measured precisely and accurately.     

Isotope ratio determination in boron analysis

Traditionally, boron (B) isotope ratios have been determined using thermal ionization mass spectrometry (TIMS) and, to some extent, secondary ion mass spectrometry (SIMS). Both TIMS and SIMS use a high-resolution mass analyzer, but differ in analyte ionization methods. TIMS uses electrons from a hot filament, whereas SIMS employs an energetic primary ion beam of Ga+, Cs+, or O- for analyte ionization. TIMS can be used in negative or positive ion modes with high sensitivity and precision of B isotope ratio determination. However, isobaric interferences may be a problem, if the sample is not well purified and/or memory of the previous sample is not removed. Time-consuming sample preparation, analyte (B) purification, and sample determination processes limit the applications of TIMS for routine analyses. SIMS can determine B and its isotope ratio in intact solid samples without destroying them, but has poorer resolution and sensitivity than TIMS, and is difficult to standardize for biological samples. Development of plasma-source mass spectrometry (MS) enabled the determination of B concentration and isotope ratio without requiring sample purification. Commonly used plasma-source MS uses an Ar inductively coupled plasma (ICP) as an ionization device interfaced to a low-resolution quadrupole mass analyzer. The quadrupole ICP-MS is less precise than TIMS and SIMS, but is a popular method for B isotope ratio determination because of its speed and convenience. B determination by ICP-MS suffers no spectroscopic interferences. However, sample matrices, memory effects, and some instrument parameters may affect the accuracy and precision of B isotope ratio determination if adequate precautions are not taken. New generations of plasma-source MS instruments using high-resolution mass analyzers provide better sensitivity and precision than the currently used quadrupole ICP-MS. Because of the convenience and high sample throughput, the high-resolution ICP-MS is expected to be the method of choice for B isotope ratio determination. The current state of instrumental capabilities is adequate for B isotope determination. However, precision and accuracy are primarily limited by sample preparation, introduction, and analytical methodology, including 1. Analyte loss and isotope fractionation during sample preparation. 2. The precision of B isotope determination in small samples, especially those containing low concentrations. 3. Difficult matrices. 4. Memory effects. Sample preparation by alkali fusion allows rapid and complete decomposition of hard-to-digest samples, but high-salt environments of the fused materials require extensive sample purification for B ratio determination. The alternative wet-ashing sample decomposition with HF also results in B loss and isotopic fractionation owing to the high volatility of BF3. Open-vessel dry- or wet-ashing methods usually do not work well for animal samples, and are also prone to B loss and contamination. Closed-vessel microwave digestion overcomes these problems, but the digests of biological materials have high C contents, which cause spectral interference on 11B and affect 11B/10B ratios. Exchange separation/preconcentration of B using exchange (cation or anion exchange, B-specific resin, e.g., Amberlite IRA-743) tend to cause B isotope fractionation, and C eluting from these resin columns may interfere with B isotope ratio determination. Memory effects of B that occur during sample determination may cause serious errors in B isotope ratio determination, especially when samples varying in B concentrations and/or isotope composition are analyzed together. Although the utilization of high-resolution plasma-source MS will undoubtedly improve analytical precision, it is the sample preparation, sample introduction, and analytical methodology that represent the primary limitation to accurate and precise B isotope ratio determination.     

Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry

This review summarizes the methods, mainly based on mass spectrometry, for the structural determination of N- and O-linked carbohydrates that are post-translationally attached to a large number of proteins and which play a key role in determining the function and biophysical properties of these compounds. Analysis of these carbohydrates has proved difficult in the past due to their structural complexity. However, modern analytical methods such as mass spectrometry have the ability to elucidate most structural details at the concentration levels required for proteomics. This review describes methods for direct examination of glycoproteins by mass spectrometry, the release of N- and O-linked glycans from glycoproteins separated in sodium dodecyl sulfate polyacrylamide electrophoresis gels, and the analysis of these compounds by techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry provides the most rapid method for comparing glycan profiles and is probably most appropriate for clinical studies. One of the most promising techniques for determining the structures of N-glycans in proteomic studies is negative ion fragmentation of electrosprayed ions. This technique combines high throughput with ease of structural interpretation and provides structural details that are difficult to obtain by classical methods.     

Electrohydrodynamic ionization mass spectrometry of biochemical materials

Electrohydrodynamic ionization mass spectrometry has been applied to a range of biochemical materials dissolved in glycerol with NaI as electrolyte. Sugars (glucose, sucrose, raffinose), nucleosides (adenosine, thymidine, uridine), a tripeptide (glutathione) and an aminocyclitol antibiotic (neomycin) have been analyzed. Unambiguous analysis of a multicomponent solution has been demonstrated. All samples yielded several quasimolecular ions involving either proton or cation attachment to clusters of sample and/or solvent molecules. Unlike other techniques such as field desorption, electrohydrodynamic ionization is not observed to cause fragmentation of sample molecules. The mass spectrometer was operated so as to analyze only those ion clusters which had not undergone decomposition processes; under these conditions, most materials are ionized with similar efficiencies if the total abundance of all characteristic quasimolecular ions is considered. Information regarding the amino acid sequence of glutathione was obtained by thermal pretreatment of the glycerol solution before mass analysis. Positive and negative ion spectra give complementary information which can resolve potential ambiguities regarding the exact composition of quasimolecular ions. Electrohydrodynamic ionization mass spectrometry should be applicable to materials which cannot be ionized by other methods.     

Stable isotope dilution gas chromatography/mass spectrometry of prostaglandins and leukotrienes

The group of arachidonic acid metabolites comprising the prostaglandins, thromboxanes, and leukotrienes (eicosanoids) are extremely potent, biologically active compounds. Their properties include proaggregatory anti-aggregatory activity for platelets, chemotactic activity for neutrophils, vasoactive activity, and contractile activity to smooth muscle. In order to determine the role of these substances in pathophysiological conditions, it is essential to have highly sensitive methods available for their analysis. It is generally accepted that combined gas chromatography/mass spectrometry is the most specific technique available for the quantitative analysis of eicosanoids. However, methods based on electron impact ionization and positive ion chemical ionization are relatively insensitive, and many investigators have preferred the use of less specific but more sensitive methods based on radioimmunoassay. We have explored the use of negative ion chemical ionization mass spectrometry to improve sensitivity coupled with capillary column chromatography to maximize specificity. Conversion of the terminal carboxyl group (present in all eicosanoids) to the pentafluorobenzyl ester derivative confers excellent electron capturing properties to the molecule. The derivative undergoes highly efficient thermal electron capture in the gas phase, and any fragmentation that occurs subsequently is directed almost entirely away from the analyte molecule. The stabilized carboxylate anion that results carries at least 30% of the total ion current. Using selected ion monitoring techniques it is possible to detect eicosanoids in the range 1–8 pg on column. This methodology has been applied to the development of stable isotope dilution assays for plasma 6-oxo-prostaglandin (PG) F_1α (1) and for the simultaneous analysis of six biologically important PGs in biological fluids (2). In addition, stable isotope dilution techniques have been developed for the analysis of serum thromboxane B₂ and serum leukotriene B₄ (3). The application of this technology to understanding the role of arachidonic acid metabolism in humans will be discussed.     

Application of gas chromatography-surface ionization organic mass spectrometry to forensic toxicology

Surface ionization (SI), which consists in the formation of positive and negative ions along the course of thermal desorption of particles from a solid surface, was first applied as a detector for gas chromatography (GC), GC-surface ionization detection (SID); we developed many new sensitive methods for the determination of abused and other drugs by GC-SID. Recently, Fujii has devised a combination of SI and a quadrupole mass spectrometer and named this system a surface ionization organic mass spectrometer (SIOMS), which is highly selective and sensitive for organic compounds containing tertiary amino groups. We have tried to apply this mass spectrometer to forensic toxicological study; so far we have succeeded in determining important drugs-of-abuse and toxic compounds, such as phencyclidine (PCP), pethidine, pentazocine, MPTP and its derivatives from human body fluids with high sensitivity and selectivity. In this review, we describe our recent studies on the application of GC-SIOMS to forensic toxicology.     

Techniques for boron determination and their application to the analysis of plant and soil samples

This paper reviews techniques for determining B concentration and isotopic ratio and their application to soil and plant samples. Boron concentration has been determined utilising spectrophotometry, potentiometry, chromatography, flame atomic emission and absorption spectrometry, inductively coupled plasma (ICP) optical emission (OES) and mass spectrometry (MS), and neutron activation analysis using neutron radiography and prompt- activation analysis. Isotopic ratios of B have been measured by ICP–MS, thermal ionisation mass spectrometry (TIMS) and secondary ion mass spectrometry (SIMS). For isotopic measurements, TIMS and SIMS are more sensitive and provide higher degrees of accuracy and resolution than ICP–MS, however, extensive sample preparation and purification, and time-consuming measurements limit their usefulness for routine analyses.While the spectrophotometric technique using a colorimetric reaction of B with azomethine-H has been the most extensively applied B determination method for soil and plant samples, colorimetric methods, in general, suffer from numerous interferences and have poor sensitivity and precision. The prompt- method can determine B concentration in intact samples which enables this method to be especially useful for some applications in agriculture. Research involving B behaviour in plant and soil environments would benefit from this technology. In recent years, the use of ICP–OES and ICP–MS for B determination in plant and soil samples has grown tremendously. The application of ICP–OES brought a significant improvement in B analysis because of its simplicity, sensitivity and multielement detection capability. However, besides matrix interferences, the two most sensitive emission lines for B suffer strong spectral interference from Fe. The ICP–OES is not adequately sensitive for some nutritional work involving low B concentrations and B translocation studies using the isotope tracer technique.Plasma is one of the most effective analyte ionisers and MS is the most sensitive ion detector. Coupling of plasma with MS resulted in the development of plasma source MS technology (ICP–MS) which has outperformed all previous analytical methods for trace element determination. Boron determination by ICP–MS suffers no spectroscopic interferences, and is considered the most practical and convenient technique for B isotope determination. The ability of ICP–MS to measure isotopic ratios as well as B concentration enables: (1) B concentration determination by the isotope dilution method, (2) verification of B concentration by isotope fingerprinting in routine analysis and (3) determination of total B concentration as well as B isotope ratio in the same run for biological tracer studies. Therefore, ICP–MS is the method of choice among the present-day technologies for determining B concentration and a convenient method for B isotope determination. In recent years, new generations of plasma-source MS instruments have been developed using alternative plasma generation methods and high-resolution mass spectrometers. These instruments are expected to bring further improvements in accuracy, sensitivity and precision of B determination.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.     

Soft ionization mass spectral methods for lipid analysis

Chemical ionization (CI), field ionization (FI) and field desorption (FD) are sometimes preferable to electron impact (EI) mass spectrometry as methods for obtaining abundant high-mass ions from lipids. FD often provides mass spectral information which is unobtainable by other methods, and is the best method for obtaining molecular weight information. Fragment ions are observed in the spectra from all the ionization methods, which provide structural information complementing that obtainable from an EI spectrum. Using CI, high-mass ions carrying a large proportion of the total ionization current can be monitored by selected ion monitoring, resulting in enhanced sensitivity for quantitative studies in some cases.     

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.     

Refining the termination age of the Cryogenian Sturtian glaciation in South China

Zhou et al. (2004) reported a zircon U-Pb isotope dilution – thermal ionization mass spectrometry (ID-TIMS) age of 662.9 ± 4.3 Ma from the basal Datangpo Formation of the Cryogenian System in South China. It was the first zircon U-Pb age to constrain both the termination of Sturtian glaciation and the onset of Marinoan glaciation, despite its much larger uncertainty expected for modern ID-TIMS analysis. In this study, we carried out U-Pb chemical abrasion – isotope dilution – isotope ratio mass spectrometry (CA-ID-IRMS) analysis on the same sample, yielding a precise ²⁰⁶Pb/²³⁸U date of 659.96 ± 0.46 Ma. Bayesian eruption age estimate indicates that the ash layer was most likely deposited at 659.23 ± 1.24 Ma. The present study suggests that the Sturtian glaciation terminated before ∼660 Ma. Together with previously reported radiometric dates, the precise U-Pb age presented herein helps refine the current Cryogenian chronology.