In‐line coupled single drop liquid–liquid–liquid microextraction with capillary electrophoresis for determining fluoroquinolones in water samples

A simple in‐line single drop liquid–liquid–liquid microextraction (SD‐LLLME) coupled with CE for the determination of two fluoroquinolones was developed. The method is capable to quantify trace amount of analytes in water samples and to improve the sensitivity of CE detection. For the SD‐LLLME, a thin layer of organic phase was used to separate a drop of 0.1 M NaOH hanging at the inlet of the capillary from the aqueous donor phase. By this way, the analytes were extracted to the acceptor phase through the organic layer based on their acidic/basic dissociation equilibrium. The drop was immersed into the organic phase during 10 min for extraction and then it is directly injected into the capillary for the analysis. Parameters such as type and volume of organic solvent phase, aqueous donor, and acceptor phases and extraction time and temperature were optimized. The enrichment factor was calculated, resulting 40‐fold for enrofloxacin (ENR) and sixfold for ciprofloxacin (CIP). The linear range were 20–400 μg/L for ENR and 60–400 μg/L for CIP. The detection limits were 10.1 μg/L and 55.3 μg/L for ENR and CIP, respectively, and a good reproducibility was obtained (4.4% for ENR and 5.6% for CIP). Two real water samples were analysed applying the new method and the obtained results presented satisfactory recovery percentages (90–100.3%).     

The formation of non-soluble complexes between polyethyleneimine-anions and their potential use to isolate enzymes

The aqueous solution behavior of polyethyleneimine (a synthetic cationic polymer) in the presence of anions with two or more electrical charges (citrate, phosphate, sulphate, malate, malonate and succinate) was studied by means of turbidimetry and light scattering. Polyethyleneimine forms non-soluble complexes with these anions, which behave as a pseudo-polyampholyte with an isoelectrical pH value dependent on the type of anion. The effect of pH, polymer concentration and ionic strength on the non-soluble complexes formation was examined. The complex precipitation pH range was between 3.5 and 8.0 and also depended on the type of anion. The complex formation was inhibited by the ionic strength in agreement with the electrostatic mechanism of the non-soluble complex formation. Model proteins with isoelectric pH from 1 to 10 were assayed in orden to be precipitated by these complexes. It was found that the non-soluble polyethyleneimine-anion complexes have the property to precipitate macromolecules charged with an opposite electrical charge.     

Influence of synthesis conditions on the cross-link architecture of silsesquioxanes prepared by in situ water production route

The esterification reaction between carboxylic acids and alcohols has been used as the source of water for the hydrolysis-condensation reactions of difunctional and trifunctional organosilanes. Diphenylsilanediol (DPDO) has been reacted with methacryloxypropyltrimethoxysilane (MPTMS) and glycidoxypropyltrimethoxysilane (GPTMS) and the obtained products have been characterized by vibrational spectroscopy (FT-IR, FT-Raman) nuclear magnetic resonance (NMR) and gel permeation chromatography. The relation between water availability from the in situ water production process (ISWP) and silsesquioxanes morphology has been evaluated in the case of DPDO/MPTMS mixtures, changing molecular features of acids and alcohols. These measurements have shown that the pK of the carboxylic acid used in the esterification reaction has a valuable influence on the silanes cross-linking ability. Acids with low pK values and heteroatoms substituents favor the silane hydrolysis and allow the growth of high molecular weight species. Using acetic acid/ethanol mixture leads to the best results for DPDO/MPTMS reaction, with a narrow distribution of silsesquioxane species. Under the same conditions, the reaction of DPDO with GPTMS produces polymeric species and only avoiding the presence of the difunctional precursor allows to limiting the silsesquioxanes species growth.     

Gemifloxacin mesylate (GFM) stability evaluation applying a validated bioassay method and in vitro cytotoxic study

The validation of a microbiological assay applying the cylinder-plate method to determine the quinolone gemifloxacin mesylate (GFM) content is described. Using a strain of Staphylococcus epidermidis ATCC 12228 as the test organism, the GFM content in tablets at concentrations ranging from 0.5 to 4.5 μg mL⁻¹ could be determined. A standard curve was obtained by plotting three values derived from the diameters of the growth inhibition zone. A prospective validation showed that the method developed is linear (r = 0.9966), precise (repeatability and intermediate precision), accurate (100.63%), specific and robust. GFM solutions (from the drug product) exposed to direct UVA radiation (352 nm), alkaline hydrolysis, acid hydrolysis, thermal stress, hydrogen peroxide causing oxidation, and a synthetic impurity were used to evaluate the specificity of the bioassay. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two validated methods. These studies demonstrate the validity of the proposed bioassay, which allows reliable quantification of GFM in tablets and can be used as a useful alternative methodology for GFM analysis in stability studies and routine quality control. The GFM reference standard (RS), photodegraded GFM RS, and synthetic impurity samples were also studied in order to determine the preliminary in vitro cytotoxicity to peripheral blood mononuclear cells. The results indicated that the GFM RS and photodegraded GFM RS were potentially more cytotoxic than the synthetic impurity under the conditions of analysis applied.     

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the “Mfold Web Server” simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of “Dinamelt Web Server”. The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors.     

Urea-, squaramide-, and sulfonamide-based anion receptors: a thermodynamic study

In this work, we compare the anion-binding capabilities of receptors 1-5, characterized by similar structures, but possessing different hydrogen-bond-donor moieties (urea, squaramide, and sulfonamide). The presence of chromophoric substituents on the receptor's skeleton allowed the determination of association constants by performing UV/Vis titrations with the investigated anions on solutions of the receptors in pure acetonitrile. Additional quantitative studies of the anion-binding properties of receptors 1-5 were performed by isothermal titration calorimetry (ITC). The experimental results indicated that 1 and 2 formed 1:1 hydrogen-bonded complexes with most of the anions investigated. In the case of receptors 3-5, the formation of the 1:1 adduct was observed only with anions of low basicity (i.e., chloride, bromide, iodide, and hydrogen sulfate). With more basic anions (i.e., acetate and dihydrogen phosphate), both spectrophotometric and ITC titrations accounted for the deprotonation of the sulfonamide group, involving the formation of the conjugated base of the receptor.     

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of the novel inhibitor of angiogenesis E-3810 in human plasma and its application in a clinical pharmacokinetic study

E-3810, 6-[[7-[(1-aminocyclopropyl)methoxy]-6-methoxy-4-quinolyl]oxy]-N-methyl-naphthalene-1-carboxamide, is a novel, potent, dual inhibitor of vascular endothelial growth factor and fibroblast growth factor receptors with antiangiogenic properties, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated to measure the drug in human plasma on the basis of simple protein precipitation with methanol after addition of deuterated E-3810 as internal standard. The method requires a small volume of sample (100 μl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise, and accurate, with overall precision, expressed as CV%, always ≤7.1%, accuracy in the range 92.7%-104.4%, and high recovery, close to 100%. The limit of detection is 0.01 ng/ml, and the lower limit of quantitation is 2.0 ng/ml. The assay was validated in the range from the lower limit of quantitation up to 500.0 ng/ml. This is the first method developed and validated for analyzing E-3810 in human plasma. The method has been successfully applied to study E-3810 pharmacokinetics in cancer patients with solid tumors who are receiving daily oral doses of the drug during the phase I trial.     

Pyridinium/urea-based anion receptor: methine formation in the presence of basic anions

The influence of the positively charged N-methylpyridinium substituent on the anion binding tendencies of urea-based receptors has been investigated by comparing molecules 1 and 2. These receptors have been studied in acetonitrile, by performing UV-vis. and (1)H NMR titrations with several anions. UV-vis. titrations have also been performed in DMSO, MeOH and CHCl(3)/CH(3)CN mixture (1/1, v/v). In the case of 1, the presence of both H-donor and H-acceptor groups (urea and pyridine, respectively) favours aggregation and the formation of dimers in the solid state. In solution, this tendency to aggregate reduces affinity for anions with respect to the similar urea-based receptor 3. The methylation of the pyridyl group of 1 leads to the pyridinium-containing receptor 2. The pyridinium positive charge enhances the acidity of urea and increases anion affinity, as evidenced by the comparison of the binding constants. Both receptors (1-2) form stable adducts with all investigated anions. However, in the case of 2, the formation of 1?:?1 adducts with basic anions, such as acetate and fluoride, is followed by a proton transfer process. Quite interestingly, deprotonation does not involve the urea group, thus preserving the 1?:?1 adduct, as demonstrated by the (1)H NMR measurements. In particular, the proton transfer process takes place at the methylene group linking the pyridinium fragment to the receptor's skeleton. (1)H NMR studies indicate the formation of a stable neutral methine species, characterised by the loss of aromaticity by the pyridyl ring. These results open new perspectives in the field of anion recognition, as receptor 2 may by applied to the monitoring of both bound anion (through the urea unit) and excess anion in solution (through the development of the yellow methine species).