Gas chromatography/surface-assisted laser desorption ionization mass spectrometry of amphetamine-like compounds.

A variety of amphetamine-like compounds were analyzed by gas chromatography/surface-assisted laser desorption ionization mass spectrometry, GC/SALDI-MS. In the SALDI method, compounds are adsorbed on a solid SALDI substrate and directly ionized from the substrate by means of a laser pulse. The interfacing of a SALDI ion source with a gas chromatograph is presented here for the first time. The end of the GC column is situated 20 mm from the silicon substrate in the vacuum of the ion source of a time-of-flight mass spectrometer, and the compounds eluted from the GC capillary are adsorbed onto the nanostructured silicon surface. The mass spectra show very low levels of background noise and no reagent ions. GC/SALDI-MS detection limits are several orders of magnitude lower than those previously reported for GC/MS analysis of amphetamine-like compounds. The extent of fragmentation is under experimental control by changing the laser fluence.

On the role of defects and surface chemistry for surface-assisted laser desorption ionization from silicon.

The generation of ions from silicon substrates in surface-assisted laser desorption ionization (SALDI) has been studied using silicon substrates prepared and etched by a variety of different methods. The different substrates were compared with respect to their ability to generate peptide mass spectra using standard liquid sample deposition. The desorption/ionization processes were studied using gas-phase analyte deposition. Mass spectra were obtained from compounds with gas-phase basicities above 850 kJmol and with molecular weights up to 370 Da. UV, VIS, and IR lasers were used for desorption. Ionization efficiencies were measured as a function of laser fluence and accumulated laser irradiance dose. Solvent vapors were added to the ion source and shown to result in fundamental laser-induced chemical and physical changes to the substrate surfaces. It is demonstrated that both the chemical properties of the substrate surface and the presence of a highly disordered structure with a high concentration of "dangling bonds" or deep gap states are required for efficient ion generation. In particular, amorphous silicon is shown to be an excellent SALDI substrate with ionization efficiencies as high as 1%, while hydrogen-passivated amorphous silicon is SALDI inactive. Based on the results, a novel model for SALDI ion generation is proposed with the following reaction steps: (1) the adsorption of neutral analyte molecules on the SALDI surface with formation of a hydrogen bond to surface Si-OH groups, (2) the electronic excitation of the substrate to form free electron/hole pairs (their relaxation results in trapped positive charges in near-surface deep gap states, causing an increase in the acidity of the Si-OH groups and proton transfer to the analyte molecules), and (3) the thermally activated dissociation of the analyte ions from the surface via a "loose" transition state.

Microbially-influenced corrosion: damage to prostheses, delight for bacteria.

In natural and man-made environments, microbial communities thrive as biofilms on living (e.g. tissue) and inanimate (e.g. plastic, metal, wood, mineral) surfaces. Biofilms are found in a wide range of aqueous habitats, including physiological fluids. Numerous types of microorganisms are able to colonize catheters, implants, prosthetics, and other medical devices manufactured from different metallic and non-metallic materials dwelling within a human body. The development of biofilm is facilitated by the production of extracellular polymeric substances (EPS). Biofilms formed on surfaces of metallic materials may alter interfacial electrochemical processes, which can lead to increased corrosion of the colonized substratum. Deterioration of metallic materials in the presence of a biofilm is termed biocorrosion or microbially-influenced corrosion (MIC). In the field of biomaterials, ""biocorrosion"" is commonly used when describing the effect of host tissue on the corrosion of implant metals and alloys. Therefore, to avoid confusion, we will here use the term MIC as a reference to biofilm-influenced corrosion. It is important to realise that although most metals are prone to microbial colonization, i.e. to biofouling, this does not imply that they are susceptible to MIC. For example, a metal such as titanium, accumulates biofilm, however, it still demonstrates excellent resistance against MIC. Corrosion is, by definition, an electrochemical process, therefore, electrochemical techniques are frequently employed to determine and measure the rate of abiotic, as well as biologically driven corrosion reactions. This communication addresses the use of electrochemical techniques for monitoring (i) biofilm formation on and (ii) MIC of implant metals and alloys.

Characterization of interlayer Cs+ in clay samples using secondary ion mass spectrometry with laser sample modification.

Ultraviolet laser irradiation was used to greatly enhance the secondary ion mass spectrometry (SIMS) detection of Cs(+) adsorbed to soil consisting of clay and quartz. Imaging SIMS showed that the enhancement of the Cs(+) signal was spatially heterogeneous: the intensity of the Cs(+) peak was increased by factors up to 100 for some particles but not at all for others. Analysis of standard clay samples exposed to Cs(+) showed a variable response to laser irradiation depending on the type of clay analyzed. The Cs(+) abundance was significantly enhanced when Cs(+)-exposed montmorillonite was irradiated and then analyzed using SIMS, which contrasted with the behavior of Cs(+)-exposed kaolinite, which displayed no Cs(+) enhancement. Exposed illitic clays displayed modest enhancement of Cs(+) upon laser irradiation, intermediate between that of kaolinite and montmorillonite. The results for Cs(+) were rationalized in terms of adsorption to interlayer sites within the montmorillonite, which is an expandable phyllosilicate. In these locations, Cs(+) was not initially detectable using SIMS. Upon irradiation, Cs(+) was thermally redistributed, which enabled detection using SIMS. Since neither the illite nor the kaolinite is an expandable clay, adsorption to inner-layer sites does not occur, and either modest or no laser enhancement of the Cs(+) signal is observed. Laser irradiation also produced unexpected enhancement of Ti(+) from illite and kaolinite clays that contained small quantities of Ti, which indicates the presence of microscopic titanium oxide phases in the clay materials.

An activated carbon substrate surface for laser desorption mass spectrometry

A method to obtain laser desorption/ionization mass spectra of organic compounds by depositing sample solutions onto a carbon substrate surface is demonstrated. The substrate consists of a thin layer of activated carbon particles immobilized on an aluminum support. In common with the porous carbon suspension samples used in previous "surface-assisted laser desorption/ionization" (SALDI) work, the mass spectra contain only a few "matrix" background ion peaks, minimizing interference with analyte ion peaks. The presence of glycerol ensured that the ion signals were stable over hundreds of laser shots. In addition, the carbon substrate surface has several advantages over the suspension samples. The use of a very thin layer of carbon significantly improves the sensitivity. Detection limits range from attomoles for crystal violet to femtomoles for bradykinin. Very little sample preparation is required as the analyte solution is simply pipetted onto the substrate surface and glycerol added. When using an alternate sample deposition method, a mass resolution for bradykinin of 1800 is achieved in linear time-of-flight mode. This is close to the resolution limit set by the detector system and above instrument specification for matrix-assisted laser desorption/ionization mass spectra.

Rapid determination of trace nitrophenolic organics in water by combining solid-phase extraction with surface-assisted laser desorption/ionization time-of-flight mass spectrometry.

A rapid technique for the screening of trace compounds in water by combining solid-phase extraction (SPE) with activated carbon surface-assisted laser desorption/ionization (SALDI) time-of-flight mass spectrometry is demonstrated. Activated carbon is used both as the sorbent in SPE and as the solid in the SALDI matrix system. This eliminates the need for an SPE elution process. After the analytes have been adsorbed on the surfaces of the activated carbon during SPE extraction, the activated carbon is directly mixed with the SALDI liquid and mass spectrometric analysis is performed. Trace phenolic compounds in water were used to demonstrate the effectiveness of the method. The detection limit for these compounds is in the ppb to ppt range.

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry.

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.

Thin-layer chromatography-mass spectrometry using activated carbon, surface-assisted laser desorption/ionization.

The analysis of compounds separated by thin-layer chromatography (TLC) by surface-assisted laser desorption ionization (SALDI) mass spectrometry has been demonstrated. The compounds are analyzed from the surface of the intact TLC plate, and the preparation of the TLC plate is rapid and robust: the gel surface is covered with 2 microns activated carbon particles, and glycerol is added. Analytes diffuse from the interior of the gel to the surface where they are adsorbed onto the activated carbon. A nitrogen laser is used to desorb analyte ions from the carbon particles in a time-of-flight mass spectrometer. A wide range of organic compounds, including peptides, can be detected, either as protonated or as cationized molecules. Interference with "matrix peaks" is limited since background TLC-SALDI mass spectra typically show only a few intense peaks at low mass. The detection limit for bradykinin from a developed plate is approximately 25 ng (calculated for S/N = 3). The mass resolution (FWHM) varied from a high of about 500 to a low of about 100. This variability was likely due to surface charging. Methods to improve both mass resolution and sensitivity of TLC-SALDI are suggested.

Infrared, surface-assisted laser desorption ionization mass spectrometry on frozen aqueous solutions of proteins and peptides using suspensions of organic solids.

Surface-assisted, laser desorption ionization (SALDI) time-of-flight mass spectra of proteins and peptides have been obtained from bulk frozen aqueous solutions by adding solid organic powders to the solutions before freezing. Abundant analyte ions were obtained with a 3.28 microgram Nd:YAG/OPO laser. 20 compounds were evaluated as solid additives, and 16 yielded protein mass spectra. Successful solids included compounds like pyrene, aspartic acid, and polystyrene. The best results were obtained with nicotinic acid and indole-2-carboxylic acid, which yielded protein mass spectra anywhere on the sample and with every laser shot. Compared with ultraviolet-matrix-assisted laser desorption ionization on the same instrument, cryo-IR-SALDI had a comparable detection limit (approximately equal to 1 micro M), a lower mass resolution for peptides, and a higher mass resolution for large proteins. Approximately 2500 cryo-IR-SALDI mass spectra were obtained from a single spot on a 0.3-mm-thick frozen sample before the metal surface was reached. About 0.1 nL of frozen solution was desorbed per laser shot. The extent of protein charging varied between the SALDI solids used. With thymine, myoglobin charge states up to MH12(+12) were observed. It is tentatively concluded that observed ions are performed in the frozen sample.

Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry.

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.

Integrated delivery systems: the future for Canadian health care reform?

Influential health care leaders have recently said that it is a misnomer to refer to the current muddle of Canadian health care services and providers as a "system." Consequently, Canadian professionals and citizens have been exposed to increasing numbers of publications recommending that a move to fully and properly Integrated Delivery Systems (IDS) could rectify many of the difficulties with which our current non-system is faced. There is, however, a skepticism that the 'IDS movement' is just another in a series of fads that will come and go, without positively impacting the health care system. In this article, the authors provide arguments for why integration is critically required, an overview of types of integration, the qualities of a fully integrated system, and a discussion of the advantages and cautions to proceeding with IDS development. They conclude with the argument that--despite potential obstacles--the possible advantages of IDSs make it vital that we pursue integration.

Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.

Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions.

Laser desorption time-of-flight mass spectra of peptides and proteins, as well as of lower molecular weight analytes, have been obtained by using a pulsed nitrogen UV laser (337 nm) to irradiate mixtures of 2-150 microns graphite particles and solutions of the analytes in glycerol. Protonated analytes as well as abundant alkali cation adducts were observed. Carbon cluster ions, Cn+, typically had a low abundance but dominated the mass spectrum at elevated laser powers. In spectra of a cytochrome c tryptic digest, all but one of the tryptic peptides were easily observed. Spectra of low molecular weight analytes dissolved in glycerol are very similar to FAB spectra of the same glycerol solution with added alkali salts. However, in many peptide and protein spectra, glycerol ion abundances are very low, and the alkali ions dominate the spectra at low mass. These spectra may correspond to wet and dry surface desorption conditions, respectively. The best spectra of the larger molecules were observed under dry conditions. In these initial experiments, we have obtained a sensitivity in the pico- to nanomole range and a mass resolution of about 300. The signal intensity is as good as that in conventional MALDI, and under optimal conditions, few background peaks appear, even at low mass.

Ion transport by viscous gas flow through capillaries.

The effects of a number of experimental parameters on the efficiency of ion transport by viscous gas flow through narrow capillaries have been studied. Both electrospray and corona ion sources were used. The experimental data are consistent with ions loss to the walls of the capillary, which initially is caused mainly by space-charge expansion, but later is caused by diffusion. These processes can result in severe discrimination against low mass ions. The extent of ion loss may be calculated by using a simple model for radial diffusional loss in long cylinders, with an exponential decay of the ion density along the transport capillary. However, such a simple model underestimates ion loss by ignoring the effects of space-charge, turbulent flow, and rapid decay of higher radial diffusion modes (enhanced loss of ions that enter the capillary close to the wall). In contrast, Monte Carlo simulations showed that the effect of the parabolic velocity profile, under laminar flow conditions, is to increase the transmitted ion current, sometimes by several orders of magnitude, relative to the predictions of the simple diffusion model. After considering all these factors, the transmitted current from a corona was well reproduced by using mobility values for ions formed in such discharges. However, the measured transmitted current from an electrospray source was much too high. To explain this, it was necessary to assume that about 2% of the electrospray current is carried by aerosol particles with radii in the 10-25-Å range. Finally, it is argued that in glass capillaries wall charging may explain why the transmitted ion current is observed to be very similar to that in metal capillaries.

Role of ion-ion recombination for alkali chloride cluster formation in liquid secondary ion mass spectrometry.

Liquid secondary ionization mass spectra of solutions of alkali chlorides in glycerol were studied as a function of salt concentration. The experimental abundances of glycerol ions and of Cs(+)(CsCl) n cluster ions were successfully reproduced by assuming that most of the randomly distributed ions pair up with counterions shortly after impact. Further, it is considered that clustering (or proton transfer) reactions occur mainly between an ion that survives the pairing process and ion pairs (or basic analytes) in the immediate vicinity; however, some mixing undoubtedly occurs in the later stages of the desorption process. At the density of the original matrix, the range of proton transfer is calculated to be 5-15 Å, and that of clustering approximately 25% shorter. These reaction distances are inversely correlated with the internal energy of the ejected ions. In general, liquid secondary ionization mass spectra of alkali chloride solutions can be seen to result from competitive ion-ion recombination reactions in the decaying matrix. Finally, from the abundances of cluster ions containing [glycerol - H](-) ions, it is estimated that approximately 1% of the glycerol molecules in the ejected volume are ionized in the collision cascade.

Postoperative diskitis: distinguishing early MR imaging findings from normal postoperative disk space changes.

To distinguish early magnetic resonance (MR) imaging findings in postoperative diskitis from normal postoperative changes, a prospective study was performed in 15 asymptomatic patients (17 disk levels) who underwent uncomplicated lumbar diskectomy and seven patients with proved postoperative diskitis. On postoperative MR images, four of the asymptomatic patients had a finding that could also be seen in patients with diskitis. Gadolinium enhancement was useful in making the distinction and occurred as follows: (a) vertebral bone marrow: all seven diskitis patients and one asymptomatic patient; (b) disk space: five diskitis patients and three asymptomatic patients; and (c) posterior anulus fibrosus: all seven diskitis patients and 13 asymptomatic patients (14 of 17 levels). This entire triad of findings, which is strongly suggestive of postoperative diskitis, was not seen in any of the asymptomatic patients. Changes in the disk space and adjacent bone marrow on pre- and post-contrast MR images after routine diskectomy are uncommon and should not be assumed to be normal postoperative changes without careful consideration and analysis for early diskitis.

Contrast-enhanced MR imaging performed after successful lumbar disk surgery: prospective study.

A prospective study was undertaken to establish the normal spectrum and timing of gadolinium-enhanced magnetic resonance (MR) imaging findings in 15 patients who had resolution of symptoms after successful lumbar disk surgery. Enhancement of the facet joints (in 88% of disk levels) and paraspinal muscles (100%) decreased gradually after surgery. Enhancement of the decompressed nerve root tracked proximally toward the conus medullaris in 62% at 3 weeks and was absent in all by 6 months postoperatively. Areas of intermediate signal intensity with peripheral enhancement and mass effect were seen on T1-weighted images at the site of the original disk herniation in 38% at 3 weeks and 12% at 3 months, despite complete relief of leg pain. These results reveal that even in successfully treated (asymptomatic) patients, residual mass effect on the neural elements may frequently simulate a recurrent or residual disk fragment. There is an orderly progression of imaging changes during the first 6 months after lumbar surgery that limits the interpretation of MR examinations during that period.

Use of femoral venous catheters in critically ill adults: prospective study.

OBJECTIVE: To determine the frequency of clinically important complications of femoral venous catheters. DESIGN: Prospective survey of major and minor complications. SETTING: A mixed medical/surgical ICU in a university hospital. PATIENTS: One hundred twenty-three patients admitted to the ICU who underwent femoral venous catheterization over a 2-yr period. MEASUREMENTS AND MAIN RESULTS: There were 150 catheters inserted in 123 patients for a mean duration of 6.4 days. There were no major complications including catheter-related sepsis. Minor complications consisted of arterial puncture (9.3%), local bleeding (10%), and local inflammation (4.7%). Critical care fellows had a significantly lower rate (6%) of insertion complications than interns or medical students (16%). We did not specifically look at the frequency of deep venous thrombosis. CONCLUSIONS: Femoral venous catheterization offers an alternative site of insertion to the subclavian and jugular veins for central venous access in the critically ill. The occurrence rate of clinically important complications is acceptably low.

Space-charge-dominated mass spectrometry ion sources: Modeling and sensitivity.

The factors determining the sensitivity of space-charge-dominated (SCD) unipolar ion sources, such as electrospray (ESP) and corona atmospheric pressure ionization (API) have been studied theoretically. The most important parameters are the ion density and ion drift time in the vicinity of the sampling orifice. These are obtained by solving a system of differential equations, "the space-charge problem." For some simple geometries, analytical solutions are known. For a more realistic "needle-in-can" geometry, a solution to the space-charge problem was obtained using a finite-element method. The results illustrate some general characteristics of SCD ion sources. It is shown that for typical operating conditions the minimum voltage required to overcome the space-charge effect in corona API or ESP ion sources constitutes a dominant or significant fraction of total applied voltage. Further, the electric field and the ion density in the region of the ion-sampling orifice as well as the ion residence time in the source are determined mainly by the space charge. Finally, absolute sensitivities of corona API ion sources were calculated by using a geometry-independent treatment of space charge.