Vilmos Kertesz

Fully automated liquid extraction-based surface sampling and ionization using a chip-based robotic nanoelectrospray platform.

A fully automated liquid extraction-based surface sampling device utilizing an Advion NanoMate chip-based infusion nanoelectrospray ionization system is reported. Analyses were enabled for discrete spot sampling by using the Advanced User Interface of the current commercial control software. This software interface provided the parameter control necessary for the NanoMate robotic pipettor to both form and withdraw a liquid microjunction for sampling from a surface. The system was tested with three types of analytically important sample surface types, viz., spotted sample arrays on a MALDI plate, dried blood spots on paper, and whole-body thin tissue sections from drug dosed mice. The qualitative and quantitative data were consistent with previous studies employing other liquid extraction-based surface sampling techniques.

Computer-Aided Design of a Sulfate-Encapsulating Receptor

Custom built: A promising new approach towards more efficient self-assembled cage receptors through computer-aided design is demonstrated. The resulting M(4)L(6) tetrahedral cage, internally functionalized with accurately positioned urea hydrogen-bonding groups (see structure; yellow: predicted, blue: experimental, space-filling: SO(4)(2-)), proved to be a remarkably strong sulfate receptor in water.

Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography.

Desorption electrospray ionization tandem mass spectrometry (DESI-MS/MS) and whole-body autoradiography (WBA) were used for chemical imaging of whole-body thin tissue sections of mice intravenously dosed with propranolol (7.5 mg/kg). DESI-MS/MS imaging utilized selected reaction monitoring detection performed on an AB/MDS SCIEX 4000 QTRAP mass spectrometer equipped with a prototype extended length particle discriminator interface. Propranolol images of the tissue sections using DESI-MS/MS were obtained at surface scan rates of 0.1, 0.5, 2, and 7 mm/s. Although signal decreased with increasing scan rate, useful whole-body images for propranolol were obtained from the tissues even at 7 mm/s, which required just 79 min of analysis time. Attempts to detect and image the distribution of the known propranolol metabolites were unsuccessful. Regions of the tissue sections showing the most radioactivity from WBA sections were excised and analyzed by high-performance liquid chromatography (HPLC) with radiochemical detection to determine relative levels of propranolol and metabolites present. Comparison of the DESI-MS/MS signal for propranolol and the radioactivity attributed to propranolol from WBA sections indicated nominal agreement between the two techniques for the amount of propranolol in the brain, lung, and liver. Data from the kidney showed an unexplained disparity between the two techniques. The results of this study show the feasibility of using DESI-MS/MS to obtain useful chemical images of a drug in whole-body thin tissue sections following drug administration at a pharmacologically relevant level. Further optimization to improve sensitivity and enable detection of the drug metabolites will be among the requirements necessary to move DESI-MS/MS chemical imaging forward as a practical tool in drug discovery.

Improved imaging resolution in desorption electrospray ionization mass spectrometry

The imaging resolution of desorption electrospray ionization mass spectrometry (DESI-MS) was investigated using printed patterns on paper and thin-layer chromatography (TLC) plate surfaces. Resolution approaching 40 microm was achieved with a typical DESI-MS setup, which is approximately 5 times better than the best resolution reported previously. This improvement was accomplished with careful control of operational parameters (particularly spray tip-to-surface distance, solvent flow rate, and spacing of lane scans). In addition, an appropriately strong analyte/surface interaction and uniform surface texture on the size scale no larger than the desired imaging resolution were required to achieve this resolution. Overall, conditions providing the smallest possible effective desorption/ionization area in the DESI impact plume region and minimizing the analyte redistribution on the surface during analysis led to improved DESI-MS imaging resolution.

Poly(methylene blue) modified electrode sensor for haemoglobin

Poly(methylene blue) modified electrodes have been prepared by potential cycling from aqueous electrolyte solution containing methylene blue on glassy carbon substrates. They have been used successfully for the determination of haemoglobin via its electrooxidation at fixed potential using the batch injection analysis technique in which whole blood samples diluted with phosphate or acetate buffer are injected directly over the modified electrode immersed in inert electrolyte. Electrode response is fast, stable, and reproducible. For blood from healthy donors, a good linear correlation was found between the results from the electrochemical injection method and the conventional clinical spectrophotometric measurement after cyanidation. The blood from potentially ill donors showed deviations from the linear correlation suggesting that this may be a good method for detecting haematological problems.

Application of a liquid extraction based sealing surface sampling probe for mass spectrometric analysis of dried blood spots and mouse whole-body thin tissue sections.

The utility of a liquid extraction based sealing surface sampling probe (SSSP) for the direct mass spectrometric analysis of targeted drugs and metabolites in dried blood spots (DBSs) and whole mouse thin tissue sections was demonstrated. The accuracy and precision for the quantitative analysis of a minimum of 50 ng/mL sitamaquine or acetaminophen in DBSs on paper were well within the required 15% dictated by internationally recognized acceptance criteria for assay validations. Analysis of whole-body mouse thin tissue sections from animals dosed with propranolol, adhered to an adhesive tape substrate, provided semiquantitative information for propranolol and its hydroxyproranolol glucuronide metabolite within specific organs of the tissue. The relative abundances recorded for the two compounds in the brain, lung, kidney, and liver were in nominal agreement with previously reported amounts based on analysis using a liquid microjunction surface sampling probe (LMJ-SSP), whole-body autoradiography (WBA), and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability to sample and analyze from tape-adhered tissue samples, which are generally employed in WBA analysis, presents the possibility of consecutive WBA and SSSP-MS analysis of the same tissue section. This would facilitate assignment, and possibly quantitation, of the different molecular forms of total drug related material detected in the WBA analysis. The flexibility to sample larger or smaller spot sizes, alternative probe sealing mechanisms, and a reduction in internal volumes and associated sample carryover issues will be among the first simple improvements necessary to make the SSSP-MS method a practical DBS and/or thin tissue section analysis tool or to expand its use to other surface sampling applications.

Liquid microjunction surface sampling coupled with high-pressure liquid chromatography-electrospray ionization-mass spectrometry for analysis of drugs and metabolites in whole-body thin tissue sections.

In this work, a commercially available autosampler was adapted to perform direct liquid microjunction (LMJ) surface sampling followed by a high-pressure liquid chromatography (HPLC) separation of the extract components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the utility of coupling a separation with this direct liquid extraction based surface sampling approach, four different organs (brain, lung, kidney, and liver) from whole-body thin tissue sections of propranolol dosed and control mice were examined. The parent drug was observed in the chromatograms of the surface sampling extracts from all the organs of the dosed mouse examined. In addition, two isomeric phase II metabolites of propranolol (an aliphatic and an aromatic hydroxypropranolol glucuronide) were observed in the chromatograms of the extracts from lung, kidney, and liver. Confirming the presence of one or the other or both of these glucuronides in the extract from the various organs was not possible without the separation. These drug and metabolite data obtained using the LMJ surface sampling/HPLC-MS method and the results achieved by analyzing similar samples by conventional extraction of the tissues and subsequent HPLC-MS analysis were consistent. The ability to directly and efficiently sample from thin tissue sections via a liquid extraction and then perform a subsequent liquid phase separation increases the utility of this liquid extraction surface sampling approach.

Electrochemical quartz crystal microbalance study of the influence of the solution composition on the behaviour of poly(aniline) electrodes

Cyclic and potentiostatic electrochemical quartz crystal microbalance measurements are reported for poly(aniline) deposited on Pt electrodes and immersed in aqueous solutions of HClO4, 4-toluenesulfonic acid and 5-sulfosalicylic acid. The effect of solution composition on the mass changes occurring simultaneously with electrochemical transformations of the polymer was investigated. The results indicate that the pH and potential-dependent relative contribution of anions and cations to the overall ion exchange process is in connection with the degree of protonation of the reduced, partially and fully oxidised forms of the poly(aniline). The small mass change at lower potentials can be rationalised in terms of a model in which it is assumed that the expulsion of protons at the beginning of the first oxidation step and the incorporation of anions at a later stage of oxidation is basically determined by thermodynamics and not the kinetics of the system. Kinetic complications also arise for several reasons such as slow diffusion of ions and solvent molecules and relaxation of the polymer network. The actual film morphology depends on the nature of the solution used. It is proved that the pore size exclusion effect in the case of larger anions does not exist as thought previously, inasmuch as the originally compact structure formed in perchlorate solution can be transformed to a more open and swollen one under continuous cycling in the solution containing electrolyte of large ions.

Electropolymerization of Methylene Blue Investigated Using On‐Line Electrochemistry/Electrospray Mass Spectrometry

The initial products of the electrochemical polymerization of methylene blue were studied using a thin-layer flow cell coupled on-line with electrospray mass spectrometry. A potentiostatic technique was applied to initiate the polymerization while the soluble n-mers generated were monitored by mass spectrometry. Methylene blue dimers and trimers were observed at m/z values consistent with either “nitrogen-to-ring” coupling of the monomers or with “ring-to-ring” coupling of demethylated monomers during the electropolymerization. Experiments accomplished in D2O-based solutions prove that “nitrogen-to-ring” coupling is the dominant process.

HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions

Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low R(f) values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation.