Polymer diffusion in high-M/low-M hard-soft latex blends

This paper describes experiments that investigate the use of low glass transition temperature (T _g) latex particles consisting of oligomer to promote polymer diffusion in films formed from high molar mass polymer latex. The chemical composition of both polymers was similar. Fluorescence resonance energy transfer (FRET) was used to follow the rate of polymer diffusion for samples in which the high molar mass polymer was labeled with appropriate donor and acceptor dyes. In these latex blends, the presence of the oligomer (with M _n = 24,000 g/mol, M _w/M _n = 2) was so effective at promoting the interdiffusion of the higher molar mass poly(butyl acrylate-co-methyl methacrylate; PBA/MMA = 1:1 by weight) polymer (with M _n = 43,00 g/mol, M _w/M _n = 3) that a significant amount of interdiffusion occurred during film drying. Additional polymer diffusion occurred during film aging and annealing, and this effect could be described quantitatively in terms of free-volume theory. This paper is dedicated to Professor Haruma Kawaguchi to honor his many contributions to the field of latex particles and their applications.     

Fast dynamic wetting of polymer surfaces by miscible and immiscible liquids

The initial stages of spontaneous spreading of a solvent drop (toluene) on the surface of a soluble polymer (polystyrene) have been studied with a high-speed camera. For drops of 1–4 μL volume, the increase in contact radius r can be described by a power law r μ t^a r \propto {t^{\alpha }} , with the spreading exponent α = 0.50 and for the first ≈8 ms. Thereafter, the three-phase contact line was pinned leading to a macroscopic static contact angle of Θ₀ = 12–15°. The insoluble liquids ethanol (α = 0.47, Θ₀ = 0) and water (α = 0.35, Θ₀ = 90°) showed a slower spreading. We attribute the fast spreading of toluene to the strong interaction with the polymer, like in reactive wetting. The finite macroscopic contact angle indicates the formation of a ridge by softening of polystyrene due to permeated toluene and the subsequent plastic deformation by the surface tension of the liquid. This interpretation is supported by experiments on polymers grafted from a silicon wafer. Toluene completely wets polymer brush surfaces. Transport of toluene through the vapor phase plays a significant role.     

Lack of effect of oral supplementation with antioxidants on cholesterol oxidation product concentration of human plasma,as revealed by an improved gas chromatography method

A gas chromatographic method was successfully applied to determine cholesterol oxidation products (COPs) in human plasma. The linearity, precision, recovery and sensitivity of the method were determined. Oral supplementation with a combination of vitamin E (800 IU), C (1 g) and β-carotene (24 mg), given for 21 days to 21 patients, did not significantly decrease plasma COP content. No correlations (n = 26) were found between initial plasma COP content and the following parameters: age, body mass index, plasma content of α-tocopherol, cholesterol, high-density lipoprotein cholesterol and triglycerides, and fat, natural antioxidant and oxidized lipid intake. Differences in plasma COP content between type 2 diabetic (n = 6) and nondiabetic (n = 20) patients were not statistically significant. The results from this study lead us to hypothesize that the nonenzymatic oxidation of cholesterol in plasma is negligible compared to COPs originating from the diet. This article also includes a comprehensive review of the drawbacks of the analytical methods of COP determination in plasma and serum.     

Characterizing ion mobility-mass spectrometry conformation space for the analysis of complex biological samples

The conformation space occupied by different classes of biomolecules measured by ion mobility-mass spectrometry (IM-MS) is described for utility in the characterization of complex biological samples. Although the qualitative separation of different classes of biomolecules on the basis of structure or collision cross section is known, there is relatively little quantitative cross-section information available for species apart from peptides. In this report, collision cross sections are measured for a large suite of biologically salient species, including oligonucleotides (n = 96), carbohydrates (n = 192), and lipids (n = 53), which are compared to reported values for peptides (n = 610). In general, signals for each class are highly correlated, and at a given mass, these correlations result in predicted collision cross sections that increase in the order oligonucleotides < carbohydrates < peptides < lipids. The specific correlations are described by logarithmic regressions, which best approximate the theoretical trend of increasing collision cross section as a function of increasing mass. A statistical treatment of the signals observed within each molecular class suggests that the breadth of conformation space occupied by each class increases in the order lipids < oligonucleotides < peptides < carbohydrates. The utility of conformation space analysis in the direct analysis of complex biological samples is described, both in the context of qualitative molecular class identification and in fine structure examination within a class. The latter is demonstrated in IM-MS separations of isobaric oligonucleotides, which are interpreted by molecular dynamics simulations. Figure Potential for performing simultaneous “omics” through the separation of biomolecular classes on the basis of structure and mass using ion mobility-mass spectrometry Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva

The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min^-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r ² = 0.998 ± 0.002 for plasma (n = 10) and r ² = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml^-1, with a limit of quantification at 0.25 μg ml^-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.     

Determination of ochratoxin A in grains by immuno-ultrafiltration and HPLC-fluorescence detection after postcolumn derivatisation in an electrochemical cell

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg^-1 and 1 μg kg^-1. The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.     

Production and Stability of Protease from <Emphasis Type="Italic">Candida buinensis</Emphasis>

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 2³ full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol).     

Enzyme-linked immunosorbent assays for the sensitive analysis of 2,4-dinitroaniline and 2,6-dinitroaniline in water and soil

The development and characterization of one rat monoclonal antibody (mAb) for 2,4-dinitroaniline and of two rat mAbs for 2,6-dinitroaniline are described. With the immunization of rats with 2,4,6-trinitrophenyl-glycylglycine–keyhole limpet hemocyanine (KLH) conjugate one mAb (PK 5H6) has been developed and formatted into a competitive enzyme-linked immunosorbent assay (ELISA). This assay no. 1 is very sensitive for 2,4-dinitroaniline with a test midpoint of 0.24 ± 0.06 μg L⁻¹ (n = 19) in 40 mM phosphate-buffered saline (PBS). A second hapten, 3-(4-amino-2,6-dinitrophenyl)propionic acid, which was also conjugated to KLH and used for the immunization of rats, led to two sensitive ELISAs for 2,6-dinitroaniline in 40 mM PBS with test midpoints of 0.61 ± 0.08 μg L⁻¹ (n = 15; mAb DNT4 3C6; assay no. 2) and 0.94 ± 0.29 μg L⁻¹ (n = 17; mAb DNT4 1A7, assay no. 3). Selectivities of all mAbs were checked with more than 20 compounds, including nitroaromatic compounds, 2,6-dinitroaniline pesticides, and other substituted derivatives of aniline. As very noticeable cross-reactivities, all mAbs recognize 2-chloro-4,6-dinitroaniline, 4-chloro-2,6-dinitroaniline and 2-bromo-4,6-dinitroaniline, the last of these being a major metabolite of the azo dye Disperse Blue 79. As first demonstrations of applications, two ELISAs (assays no. 1 and 2) were used for the analysis of 2,4- or 2,6-dinitroaniline in spiked water and soil samples. Recovery data were determined and the majority of these data were in the range of 90–120%. These assays can contribute to a very cost-effective and environmentally friendly immunochemical surveillance monitoring of environmental samples for contaminations with these compounds. To the best of the authors’ knowledge, these are the first antibodies described for 2,4-dinitroaniline and for 2,6-dinitroaniline.     

Accelerated sample treatment for screening of banned doping substances by GC–MS: ultrasonication versus microwave energy

A comparison between ultrasonication and microwave irradiation as tools to achieve a rapid sample treatment for the analysis of banned doping substances in human urine by means of gas chromatography–mass spectrometry (GC–MS) was performed. The following variables were studied and optimised: (i) time of treatment, (ii) temperature, (iii) microwave power and (iv) ultrasonic amplitude. The results were evaluated and compared with those achieved by the routine method used in the World Anti-Doping Agency (WADA) accredited Antidoping Laboratory of Rome. Only under the effect of the ultrasonic field was it possible to enhance the enzymatic hydrolysis reaction rate of conjugated compounds. Similar reaction yield to the routine method was achieved after 10 min for most compounds. Under microwave irradiation, denaturation of the enzyme occurs for high microwave power. The use of both ultrasonic or microwave energy to improve the reaction rate of the derivatisation of the target compounds with trimethyliodosilane/methyl-N-trimethylsilyltrifluoroacetamide (TMSI/MSTFA/NH₄I/2-mercaptoethanol) was also evaluated. To test the use of the two systems in the acceleration of the reaction with TMSI, a pool of 55 banned substances and/or their metabolites were used. After 3 min of ultrasonication, 34 of the 55 compounds had recoveries similar to those obtained with the classic procedure that lasts for 30 min (Student’s t test, n = 5), 18 increased to higher silylation yields, and for the compounds 13β,17α-diethyl-3α,17β-dihydroxy-5α-gonane (norboletone metabolite 1), metoprolol and metipranolol the same results were obtained increasing the ultrasonication time to 5 min. Similar results were obtained after 3 min of microwave irradiation at 1,200 W. In this case, 30 of the 55 compounds had recoveries similar to the classic procedure (Student’s t test, n = 5) whilst 18 had higher silylation yields. For the compounds 3α-hydroxy-1α-methyl-5α-androstan-17-one (mesterolone metabolite 1), 17α-ethyl-5β-estrane-3α,17β,21-triol (norethandrolone metabolite 1), epioxandrolone, 4-chloro-6β,17β-dihydroxy-17α-methyl-1,4-androstadien-3-one (chlormetandienone metabolite 1), carphedon, esmolol and bambuterol the same results were obtained after 5 min under microwave irradiation.     

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)     

Development of a highly precise ID-ICP-SFMS method for analysis of low concentrations of lead in rice flour reference materials

Microwave digestion and isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-SFMS) has been applied to the determination of Pb in rice flour. In order to achieve highly precise determination of low concentrations of Pb, the digestion blank for Pb was reduced to 0.21 ng g⁻¹ after optimization of the digestion conditions, in which 20 mL analysis solution was obtained after digestion of 0.5 g rice flour. The observed value of Pb in a non-fat milk powder certified reference material (CRM), NIST SRM 1549, was 16.8 ± 0.8 ng g⁻¹ (mean ± expanded uncertainty, k = 2; n = 5), which agreed with the certified value of 19 ± 3 ng g⁻¹ and indicated the effectiveness of the method. Analytical results for Pb in three brown rice flour CRMs, NIST SRM 1568a, NIES CRM 10-a, and NIES CRM 10-b, were 7.32 ± 0.24 ng g⁻¹ (n = 5), 1010 ± 10 ng g⁻¹ (n = 5), and 1250 ± 20 ng g⁻¹ (n = 5), respectively. The concentration of Pb in a candidate white rice flour reference material (RM) sample prepared by the National Metrology Institute of Japan (NMIJ) was observed to be 4.36 ± 0.28 ng g⁻¹ (n = 10 bottles). Figure Digestion blank of Pb was carefully reduced to approximately 0.2 ng g^-1 which permitted the highly precise determination of Pb at low ng g^-1 level in foodstuff samples by ID-SFMS     

Bioactive Electroconductive Hydrogels: The Effects of Electropolymerization Charge Density on the Storage Stability of an Enzyme-Based Biosensor

Electrode-supported hydrogels were conferred with the biospecificity of enzymes during the process of electropolymerization to give rise to a class of bioactive, stimuli-responsive co-joined interpenetrating networks of inherently conductive polymers and highly hydrated hydrogels. Glucose responsive biotransducers were prepared by potentiostatic electropolymerization [750 mV vs. Ag/AgCl (3 M KCl)] of pyrrole at Poly(hydoxyethyl methacrylate)-based hydrogel-coated Pt micro-electrodes (Φ = 100 μm) from aqueous solutions of pyrrole and glucose oxidase (GOx; 0.4 M pyrrole, 1.0 mg/ml GOx) to 1.0 and 10.0 mC/cm². Polypyrrole was them over-oxidized by cyclic voltammetry (0–1.2 V vs. Ag/AgCl, 40 cycles in PBKCl, pH = 7.0). Biotransducers were stored at 4 °C in PBKCl for up to 18 days. Amperometric dose–response at 0.4 V vs. Ag/AgCl followed by Lineweaver–Burk analysis produced enzyme kinetic parameters as a function of electropolymerization charge density and storage time. Apparent Michaelis constant (K _Mapp) increased from 18.6–152.0 mM (1.0 mC/cm²) and from 2.7–6.1 mM (10.0 mC/cm²). Biotransducer sensitivity increased to 21.2 nA/mM after 18 days and to 12.8 pA/mM after 10 days for the 1.0 and 10.0 mC/cm² membranes, respectively. Maximum current, I _max, also increased over time to 2.7 nA (1.0 mC/cm²) and to 170 pA (10.0 mC/cm²). Electropolymerization of polypyrrole is shown to be an effective means for imparting bioactivity to a hydrogel-coated microelectrode. GOx was shown to be stabilized and to increase activity over time within the electroconductive hydrogel.     

Determination of butoxyacetic acid (biomarker of ethylene glycol monobutyl ether exposure) in human urine candidate reference material

Ethylene glycol monobutyl ether (EGBE), an industrial solvent, is absorbed by the body not only by inhalation but also by dermal absorption (liquid or vapour). EGBE is metabolized to butoxyacetic acid (BAA). Pooled freeze-dried urine candidate reference material (RM) was prepared from urine obtained from persons occupationally exposed to EGBE. This material has the advantage of containing butoxyacetic acid in both the free and conjugated (glutamine and glycine) forms, as found in native urine. In all GC method modifications used, acid hydrolysis was used to release BAA from its conjugated form. The amount of butoxyacetic acid in homogeneity and stability testing was measured by GC after derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Detection was by MS in EI mode, in the authors’ laboratory. For interlaboratory comparison of the reference material GC methods with MS, FID, and ECD were used. Different extraction solvents (dichloromethane–isopropanol 2:1, ethyl acetate, or dichloromethane) and derivatisation reagents (trimethylsilyldiazomethane, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide) were used. Using ANOVA (at the statistical level α = 0.05) no changes were found in the concentration of butoxyacetic acid during fifteen month isochronous stability testing, or in homogeneity testing. The uncertainty contributions were u _h = 8.8 mg L⁻¹ and u _s = 6.5 mg L⁻¹. The concentration of butoxyacetic acid in freeze-dried urine RM was evaluated from the results of eight laboratory data sets within an interlaboratory comparison by use of the interactive statistical software IPECA. The contribution to total uncertainty derived from interlaboratory comparison was u _i = 12.7 mg L⁻¹. The reference value (c = 273 ± 33 mg L⁻¹) is an unweighted arithmetic average of accepted results. The value is traceable to the pure butoxyacetic acid (98% w/w; Acros Organic #257760010) used as calibrant. The uncertainty given is combined expanded uncertainty derived from the results from interlaboratory comparison, and from homogeneity and stability tests (k = 2). The reference material will be used to verify method performance in the biological monitoring of occupational exposure to EGBE.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

Fluctuation in the ergosterol and deoxynivalenol content in barley and malt during malting process

This paper describes determination of the deoxynivalenol and ergosterol in samples from different varieties of barley and, consequently, malt produced from this barley. In total, 20 samples of barley and 20 samples of barley malt were analyzed. The alkaline hydrolysis with consequent extraction into hexane was applied to obtain the ergosterol from cereals. Extraction to acetonitrile/water and subsequent solid-phase extraction (SPE) were used for deoxynivalenol. The determination of the samples was performed on high-performance liquid chromatography using UV detection (ergosterol) and mass spectrometric detection (deoxynivalenol). The influence of the malting process on the production of two compounds of interest was assessed from obtained results. Ergosterol concentration ranged 0.88–15.87 mg/kg in barley and 2.63–34.96 mg/kg in malt, where its content increased to 95% compared to samples before malting. The malting process was observed as having a significant effect on ergosterol concentration (P = 0.07). The maximum concentration of deoxynivalenol was found to be 641 μg/kg in barley and 499 μg/kg in malt. Its concentration was lower than the legislative limit for unprocessed cereals (1,250 μg/kg). The statistic effect of the malting process on deoxynivalenol production was not found. Linear correlation between ergosterol and deoxynivalenol content was found to be very low (barley R = 0.02, malt R = 0.01). The results revealed that it is not possible to consider the ergosterol content as the indicator of deoxynivalenol contamination of naturally molded samples.     

Solid state electrochemical of the erbium hexacyanoferrate-modified carbon ceramic electrode and its electrocatalytic oxidation of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine

A kind of erbium hexacyanoferrate (ErHCF)-modified carbon ceramic electrodes (CCEs) fabricated by mechanically attaching ErHCF samples to the surface of CCEs derived from sol–gel technique was proposed. The resulting modified electrodes exhibit well-defined redox responses with the formal potential of +0.215 V [vs saturated calomel electrode (SCE)] at a scan rate of 20 mV s⁻¹ in 0.5 M KCl (pH 7) solution. The voltammetric characteristics of the ErHCF-modified CCEs were investigated by voltammetry. Attractively, the ErHCF-modified CCEs presented good electrocatalytic activity with a marked decrease in the overvoltage about 400 mV for l-cysteine oxidation. The calibration plot for l-cysteine determination was linear at 5.0 × 10⁻⁶–1.3 × 10⁻⁴ M with a linear regression equation of I(A) = 0.558 + 0.148c (μM) (R ² = 0.9989, n = 20), and the detection limit was 2 × 10⁻⁶ M (S/N = 3). At last, the ErHCF-modified CCEs were used for amperometric detection of l-cysteine in real samples.     

His6-OPH enzyme-based bio-hybrid material for organophosphate detection

In this work, we report on the development of a bio-sensing film for the detection of organophosphorous compounds using sol–gel technology. A novel sol–gel immobilization method employing tetraethoxysilane/3-glycidoxypropyltrimethoxysilane/water hybrid material was developed and used to immobilize the hexahistidine-tagged organophosphorous hydrolase enzyme (His₆-OPH). Bio-sensing layers with encapsulated His₆-OPH of various structures (water/silane, precursor ratios) have been prepared. The optimal (P = 5:1, R = 188) bio-sensing layers retained 90% of the initial enzyme activity. Furthermore, the bio-sensing layer prepared by this method was able to maintain its activity at or above 80% of its initial activity for 2 weeks. The bio-hybrid film also showed excellent reusability and improved activity at neutral pH in comparison to the same enzyme in solution.     

Evaluation of the oxidative status of virgin olive oils with different phenolic content by direct infusion atmospheric pressure chemical ionization mass spectrometry

Atmospheric pressure chemical ionization mass spectrometry was used to predict the oxidative status of virgin olive oils (VOO) during their storage. VOO samples, with and without phenolic compounds, were stored in the dark at 60 °C up to 7 weeks. The VOO samples were diluted in an alkaline propanol/methanol mixture and directly infused into an ion-trap mass spectrometer. The abundances of the [M−H]⁻ peaks of free fatty acids, oxidized fatty acids, tocopherols and phenolic compounds, jointly with their oxidized forms, were measured and used as predictors. Two linear discriminant analysis (LDA) models were constructed in order to classify samples according to their oxidative levels. The first model was constructed using both VOO samples (with and without phenols), considering as predictors only fatty acids and their oxidized products. The second LDA model was constructed with the VOO sample with phenolic compounds considering as predictors all the peaks measured. In both models, the samples divided in the eight different storage times were correctly classified (100%) by leave-one-out cross-validation with an excellent resolution among all the category pairs (for the first model Wilks’ lambda, λ _w = 0.229 and for the second λ _w = 0.928). This method is a very fast tool for on-line monitoring of VOO oxidation status.     

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 μg serotonin/g creatinine (Cr). The limit of quantification was 4.7 μg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS − 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at −20 °C. The established reference range for serotonin was 54–366 μg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 μg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 μg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.