TOF-SIMS imaging of chlorhexidine-digluconate transport in frozen hydrated biofilms of the fungus Candida albicans

The diffusion of the anti-microbial chlorhexidine digluconate (CHG) has been studied in C. albicans biofilms by time-of-flight secondary-ion mass spectrometry (TOF-SIMS). C. albicans has been shown to become resistant to common anti-microbial agents, including CHG, when growing as a biofilm. Mass transport resistance within biofilms has commonly been suggested as a resistance mechanism, but measurement of transport for most anti-microbial agents in biofilms has proven extremely difficult because of the heterogeneity of the biofilms and the difficulty in detecting these agents within an intact biofilm. In this study, TOF-SIMS has been used to study the transport of CHG and glucose in a frozen hydrated biofilm. The TOF-SIMS images reveal a progression of CHG from the top of the biofilm to its base with time. Images suggest that there are channels within the biofilm and show preferential binding of CHG to cellular components of the biofilm. Additionally, both living and dead cells can be identified in the TOF-SIMS images by the sequestration of K⁺ and the presence of cell markers. This study demonstrates that TOF-SIMS has the unique potential to simultaneously observe the presence of an antimicrobial agent, concentration of nutrients, and the viability of the cell population.     

Method of immobilization of carboxymethyl-dextran affects resistance to tissue and cell colonization

Coatings from carboxymethylated dextrans (CMDs) were fabricated, analyzed by XPS, and investigated for their ability to inhibit corneal epithelial tissue outgrowth and bovine corneal epithelial cell attachment and growth. CMDs with differing degrees of carboxymethyl substitution and various molecular weights were synthesized by the solution reaction of dextrans with bromoacetic acid under different reactant ratios. The CMD compounds thus obtained were attached onto aminated surfaces produced in two ways: by the plasma deposition of a coating from n-heptylamine vapour, and by the plasma deposition of an acetaldehyde coating onto whose surface aldehyde groups the polyamine compounds polylysine, polyethyleneimine and polyallylamine were immobilized to provide platforms for CMD immobilization. XPS spectra showed that the latter route produced thicker coatings than the former approach. CMD molecules attached directly onto the plasma-fabricated amine surface supported some tissue migration; the extent of carboxymethyl substitution and the molecular weight of the CMDs had little influence. For CMDs immobilized via polyamine spacers, on the other hand, tissue outgrowth was completely inhibited, and again there were no discernible effects from the extent of carboxymethyl substitution and the molecular weight of the CMDs. In assays involving cell attachment and growth, analogous observations were found. Thus, the mode of immobilization of these polysaccharide coatings is the dominant factor in their anti-fouling performance, suggesting that optimization of the architecture of polysaccharide coatings may be an important factor for maximizing their cell-repellent abilities.     

The synthesis of diastereo- and enantiomerically pure beta-aminocyclopropanecarboxylic acids

The synthesis of diastereo- and enantiomerically pure beta-aminocyclopropanecarboxylic acids (beta-ACCs) is described. Starting from pyrrole, (rac)-4 is readily obtained, which was kinetically resolved by enzymatic hydrolysis. Subsequent oxidation of (-)-4 and deformylation gives rise to the cis-beta-ACC derivative (ent)-9, while (+)-10 was converted to the trans-beta-ACC derivative 8. Both 8 and (ent)-9 and their benzyl esters 13 and 16, being conformationally restricted beta-alanine or gamma-aminobutyric acid (GABA) derivatives, represent useful building blocks for peptides containing unnatural amino acids.     

The determination of biurea: a novel method to discriminate between nitrofurazone and azodicarbonamide use in food products

Recently doubts have arisen on the usefulness of semicarbazide as marker residue for the illegal use of the antibiotic nitrofurazone (NFZ) in aquaculture and poultry production. Most notably azodicarbonamide (ADC) has been implicated as an alternative source of semicarbazide. ADC is used in some countries as a dough conditioner at concentrations up to 45 mg kg(-1). The use of ADC-treated flour or dough in coated or breaded food products may generate false non-compliant results in the analytical method for nitrofurazone metabolites, which is currently in use. During the dough preparation process ADC is largely reduced to biurea, which can be considered as an appropriate marker residue of ADC. Thus far no methods have been published for the determination of biurea in food commodities. Due to its polar nature it is very difficult to generate sufficient retention on conventional C18 HPLC columns. With a TSK amide HILIC type column good retention was obtained. A straightforward extraction-dilution protocol was developed. Using a mixture of dimethyl formamide and water biurea was nearly quantitatively extracted from a variety of fresh, coated and processed products. Mass spectrometric detection was performed with positive electrospray ionisation. The sensitivity and selectivity of the mass spectrometer for biurea was very good, allowing detection at concentrations as low as 10 microg kg(-1). However, in some extracts severe ion suppression effects was observed. To overcome the implications of ion suppression on the quantitative performance of the method an isotopically-labelled biurea internal standard was synthesized and incorporated in the method. The method developed can be used effectively in nitrofurazone analysis to eliminate the risk of false non-compliant results due to the presence of azodicarbonamide-treated components in the food product.     

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Introduction of a cryosectioning-ToF-SIMS instrument for analysis of non-dehydrated biological samples

Tof-SIMS and laser-SNMS are becoming increasingly important tools for analysing the elemental and molecular distribution in biological samples. We have developed the prototype of an in-vacuum cryosectioning instrument directly attached to a ToF-SIMS/laser-SNMS instrument, which allows preparing and analyzing frozen non-dehydrated biological samples. Due to unavoidable effects during the handling, storing, and preparation of frozen samples, even inside the vacuum, it must be ensured that the analyzed surface is from the sample and not a preparation artefact. To this effect, a model structure, similar to the biological samples and with a defined chemical composition, was analyzed under varying thermal treatment before and during analysis, respectively, in order to identify a balance between evaporating and subliming adsorbed materials and the effect of freeze-drying on the measured signal.     

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.     

Energy spectra for decaying 2D turbulence in a bounded domain

We use results derived in the framework of the replica approach to study the liquid-glass thermodynamic transition. The main results are derived without using replicas and applied to the study of the Lennard-Jones binary mixture introduced by Kob and Andersen. We find that there is a phase transition due to the entropy crisis. We compute both analytically and numerically the value of the phase transition point T(K) and the specific heat in the low temperature phase.     

Quantitation of 5-fluorouracil (5-FU) in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

5-Fluorouracil (5-FU) has long had a place in the treatment of many malignancies. 5-FU plasma concentrations have been correlated with toxicity and efficacy, and therapeutic drug monitoring has been reported to result in an improved response/toxicity balance. We report validation, according to FDA guidelines, of a hydrophilic interaction chromatography (HILIC) liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of 5-FU in human plasma. The assay employed an isotopically labeled 5-FU internal standard and ethyl acetate extraction. Separation was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile/water (97:3, v/v), followed by a wash. Detection consisted of electrospray, negative-mode ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The accuracy was 96.0-102.2%, and precision was 2.1-7.5% in the concentration range of 10-10 000 ng/mL. Recovery from plasma was 46.0-72.6%, and ion suppression was 9.8-25.7%. Plasma freeze/thaw stability was 87.5-104.3%, and stability for 4 h at room temperature was 98.7-100.0%. This assay is currently being used to quantitate 5-FU in human plasma samples.     

Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A number of anticancer drugs are cytidine analogues that undergo metabolic deactivation catalyzed by cytidine deaminase (CD). 3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD, by acting as a transition-state analogue of its natural substrate cytidine. However, to date its pharmacokinetic properties have not been fully characterized, which has impaired its optimal preclinical evaluation and clinical use. We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of THU in mouse plasma. Validation was performed according to FDA guidelines. The assay employed deuterated THU as the internal standard and an acetonitrile protein precipitation step. Separation, based on hydrophilic interaction chromatography, was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water followed by a wash. Chromatographic separation was followed by positive-mode electrospray ionization MS/MS detection in the multiple reaction monitoring (MRM) mode. The assay was accurate (92.5-109.9%) and precise (2.1-9.0%) in the concentration range of 0.2-50 microg/mL. Recovery from plasma was near-complete (92.9-119.3%) and ion suppression was negligible (-17.5 to -0.2%). Plasma freeze/thaw stability (93.1-102.1%), stability for 3 months at -80 degrees C (99.5-110.9%), and stability for 4 h at room temperature (92.1-102.4%) were all in order. This assay is currently being used to quantitate THU in ongoing pharmacokinetic studies. In addition, the assay is expected to be a useful tool in any future studies involving co-administration of THU with cytidine analogues.