Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.     

One-step conversion of n-butane to isobutene over H-beta supported Pt and Pt,M (M = Cu, In, Sn) catalysts: An investigation on the role of the second metal

The one-step transformation of n-butane to isobutene was studied over H-beta zeolite supported Pt and Pt,M (M = Cu, In, Sn) catalysts. Catalytic performance of monometallic Pt/H-beta samples resulted to be affected by the support acidity, a lower number of acid sites leading to higher iso- and n-butenes selectivities and lower by-products formation. Addition of Cu, In or Sn to Pt enhanced both isobutene and n-butenes selectivities, which were in the order: Pt,In > Pt,Sn  Pt,Cu > Pt. All Pt,M samples exhibited also a higher stability than the corresponding monometallic Pt samples, the sequence of deactivation rates being: Pt > In,Pt > Cu,Pt ≈ Sn,Pt. On the basis of characterization results it was stated that the addition of Cu, In or Sn to Pt affects the n-butane dehydroisomerization modifying both the surface structure of Pt clusters and the support acidity. In particular the observed order of isobutene selectivity was related to the degree of Pt–M interaction leading to a dilution of Pt clusters, which inhibits hydrogenolysis reactions and enhances dehydrogenation processes. The decrease in the number of acid sites caused by addition of the second metal was instead accounted for the improved resistance to deactivation of Pt,M catalysts.     

Characterization of the local structures of Ti-MCM-41 and their photocatalytic reactivity for the decomposition of NO into N2 and O2

Ti-MCM-41 mesoporous molecular sieves were prepared at ambient temperature and were characterized by X-ray absorption near-edge structure and extended X-ray absorption fine structure, UV-vis, Fourier transform infrared spectroscopy, and photoluminescence spectroscopic analyses. It was found that an increase in the Ti content caused the structure of the Ti-oxides in Ti-MCM-41 to change from an isolated tetrahedral coordination to adjacent Ti-oxide species with Ti4+ of tetrahedral coordination. The photocatalytic reactivity of these catalysts for the decomposition of NO into N2 and O2 was found to strongly depend on the local structure of the Ti-oxide species including their coordination and distribution, i.e., the charge transfer excited state of the highly dispersed isolated tetrahedrally coordinated Ti-oxides act as the active sites for the photocatalytic decomposition of NO into N2 and O2.     

Development of a microparticle-based on-site immunoassay for the detection of atrazine in soil and water samples

Atrazine is widely used as a herbicide in agriculture and has been identified as a major groundwater contaminant in the US. Because of the possible hazard associated with its usage, there is a need for an efficient and economic screening method for on-site field testing of atrazine and other s-triazine herbicides in soil and water. We have developed a rapid, on-site test for the detection of atrazine based on the principle of microparticle agglutination inhibition immunoassay. The test detects 50 microg kg(-1) (0.050 ppm) atrazine in soil samples with direct extraction and 1.0 microg L(-1) atrazine in water samples when coupled with solid phase extraction.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.     

2,5-Diamidofuran anion receptors

3,4-Diphenylfuran-2,5-dicarboxylic acid bis-N-phenylamide 1 and 3,4-biphenyl-furan-2,5-dicarboxylic acid bis-N-butylamide 2 have been synthesised and shown to act as fluoride selective anion receptors in DMSO-d₆/0.5% water solution.     

Distribution of low-density lipoprotein-bound low-molecular-weight thiols: a new analytical approach

We have recently demonstrated that low-density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5-iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and -SH free groups of proteins.     

Determination of the antidepressant mirtazapine and its two main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N-desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ = 290 nm and monitoring emission at λ = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5-150 ng mL⁻¹ for mirtazapine and of 2.5-75.0 ng mL⁻¹ for N-desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL⁻¹ and the limit of detection (LOD) was 1.25 ng mL⁻¹ for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.     

Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-based chiral stationary phase

Enantiomeric separation of mecoprop, dichlorprop, and fenoprop herbicides in their acid form, commonly used to control the growth of broad-leaved weeds, was carried out by nano-liquid chromatography (nano-LC) at a flow rate of 60 nL/min, using a packed capillary column with vancomycin-modified silica particles of 5 microm. The length of chiral stationary phase was 21 cm, while the total and effective lengths were 43 and 33cm, respectively. Inner diameter was 0.075 mm. Separated peaks were detected at 195 nm. Several mixtures of methanol, water, and 500 mM ammonium acetate buffer at different pH's were tested as mobile phase, and experimental parameters such as resolution (Rs), capacity factor (k), efficiency (N/m), and enantioselectivity factor (alpha) were measured under all the test conditions. Baseline enantiomeric separation was obtained for the three studied herbicides with alpha in the range 1.6-1.9, using as the mobile phase aqueous solutions containing 85% methanol, 5% of 500mM ammonium acetate pH4.5 buffer, and 10% water. Experimental results show that the vancomycin stationary phase presents a great enantiorecognition capability towards chlorophenoxy acid herbicides on using nano-LC.     

Origins of selectivity for the [2+2] cycloaddition of alpha,beta-unsaturated ketones within a porous self-assembled organic framework

This article studies the origins of selectivity for the [2+2] cycloadditions of alpha,beta-unsaturated ketones within a porous crystalline host. The host, formed by the self-assembly of a bis-urea macrocycle, contains accessible channels of approximately 6 A diameter and forms stable inclusion complexes with a variety of cyclic and acyclic alpha,beta-unsaturated ketone derivatives. Host 1 crystals provide a robust confined reaction environment for the highly selective [2+2] cycloaddition of 3-methyl-2-cyclopentenone, 2-cyclohexenone, and 2-methyl-2-cyclopentenone, forming their respective exo head-to-tail dimers in high conversion. The products are readily extracted from the self-assembled host and the crystalline host can be efficiently recovered and reused. Molecular modeling studies indicate that the origin of the observed selectivity is due to the excellent match between the size and shape of these guests to dimensions of the host channel and to the preorganization of neighboring enones into favorable reaction geometries. Small substrates, such as acrylic acid and methylvinylketone, were bound by the host and were protected from photoreactions. Larger substrates, such as 4,4-dimethyl-2-cyclohexenone and mesityl oxide, do not undergo selective [2+2] cycloaddition reactions. In an effort to understand these differences in reactivity, we examined these host-guest complexes by thermogravimetric analysis (TGA), NMR, powder X-ray diffraction (PXRD) and molecular modeling.