Proficiency testing schemes for the assessment of Legionella PCR methodologies

Standard operating procedures used for the detection of bacteria in environmental samples are primarily based on bacterial growth on specific culture media and confirmation by biochemical and/or immunological tests. In the case of Legionella, isolation on BCYE-?? medium is the standard method, although it presents a number of drawbacks, and for this reason, the implementation of molecular methods, mainly those based on PCR, has increased over the last years. Following the ISO/IEC 17025, laboratories need an external evaluation of their work to assure the quality of the results they are producing, and the participation in proficiency testing (PT) schemes is compulsory. For those water-testing laboratories using PCR methods for Legionella, we have developed a PT scheme accredited according to ISO/IEC 17043. The preparation and the statistical analysis of the results are performed following this standard and the ISO 13528. The used samples have a very rapid and easy to use format, consisting of tablets with inactivated freeze-dried Legionella cells or freeze-dried Legionella DNA. In this PT scheme, participants evaluate both Legionella pneumophila and Legionella spp. detection systems and control the whole PCR process from the water sample concentration until the PCR results.     

Effects of medium on decarboxylation kinetics: 3-carboxybenzisoxazoles and their potential use as environmental probes in biochemistry

The decarboxylation rate of the tetramethylguanidinium salt of 3-carboxy-6-nitrobenzisoxazole in 24 pure solvents and 36 dimethyl sulfoxide binary mixtures with diglyme, acetonitrile, benzene, dichloromethane, chloroform, and methanol was analyzed in the light of the SPP, SA, and SB pure solvent scales. The results allow one to rationalize the high sensitivity of this kinetics to the reaction medium and to assess the potential use of this compound as a probe in biochemical environments. The natural environment for comparison of this kinetics was found to be the gas phase rather than the aqueous medium. In the latter, the process is much faster owing to such high polarity, which, however, is strongly diminished by the high acidity of the medium. Based on our calculations, the rate constant for the decarboxylation kinetics in the gas phase must be in the region of 2 x 10(-10) s(-1) (i.e., 3 orders of magnitude smaller than in water).     

Application of dried spot cards as a rapid sample treatment method for determining hydroxytyrosol metabolites in human urine samples. Comparison with microelution solid-phase extraction

Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3′-O-glucuronide, hydroxytyrosol-4′-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4′-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80 %, and the matrix effect (%ME) was less than 8 %. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70 % and lower than 17 %, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.     

Compounds with π(loc) → π*(deloc) electronic transitions and their solvatochromism

Molecular structures possessing atomic sites that contribute a non‐bonding electron pair to their π system (e.g. nitrogen atoms with sp² hybridization in pyrroles and anilines) usually exhibit a first absorption band whose solvatochromism is, surprisingly, sensitive only to the polarizability of the medium even though they are dipolar. As shown here, this solvatochromic behavior is a result of the first electronic transition in these compounds occurring from a substantially localized π orbital to a substantially delocalized π* orbital in the molecular structure. The high electronic delocalization present leads to a marked bathochromic band shift as the polarizability of the medium increases. It is especially relevant that this solvatochromism, which is because of the polarizability of the medium, explains the spectral shift that is only because of the redistribution of the electrons of the solvent molecules. It is important to take into account that this electronic redistribution happens instantaneously in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

On the photophysics of all-trans polyenes: hexatriene versus octatetraene

The disparate photophysical behavior of trans-1,3,5-hexatriene (nonfluorescent) and trans-1,3,5,7-octatetraene (with two fluorescence emissions) in the gas phase is explained in terms of the tendency of their 1B(u) excited states to rotate about their terminal carbon-carbon single bonds in order to adopt a quasiplanar molecular form of lower energy than the 1B(u) state in the parent all-trans structure. The origin of their disparate photophysical behavior is that such a transformation is subject to a small energy barrier in octatetraene; the barrier produces two minima (two fluorescence emissions) in the corresponding potential-energy curve. Instead of an energy barrier, hexatriene gives a 1,3-diene species which falls to the ground state so rapidly that no emission is produced.     

Solid-phase reactors as high stability reagent sources in flow analysis: selective flow injection spectrophotometric determination of cysteine in pharmaceutical formulations

The flow injection spectrophotometric determination of cysteine was carried out by reaction with cobalt(II) ions entrapped in a polymeric material and filling a packed-bed reactor; the released cobalt(II) complexed with the amino acid was monitored at 360 nm. The method worked with a high repeatability, even with independent reactors, days and solutions. Selectivity of the procedure was tested with twenty different foreign compounds found in pharmaceutical formulations containing cysteine, parent amino acids included; no serious interferences were observed. The calibration graph for cysteine was linear over the range 1-90 micrograms ml-1 with a relative standard deviation of 0.8% at 60 micrograms ml-1 (n = 158). The calculated sample throughput was 90 h-1. The method was applied to determine the content of cysteine in pharmaceutical formulations.     

A tandem-flow assembly for the chemiluminometric determination of hydroquinone

A direct chemiluminescent procedure for determination of hydroquinone based on the emergent flow methodology known as multicommutation or tandem-flow is presented for first time. The manifold was based on a set of three channels and three solenoid valves; and, the determination was performed at 60 °C and at flow-rate of 7.5 ml min⁻¹. The complete cycle lasted 35 s, which resulted in a sample flow trough of 103 h⁻¹. The chemical process was the hydroquinone oxidation with the system sulphuric acid-potassium permanganate; and the light emission was clearly enhanced by the presence of quinine sulphate and benzalkonium chloride reaching a detection limit of 30 μg l⁻¹. The dynamic interval was over the range 0.1-15.0 mg l⁻¹ and a large list of interferents were assayed; the chemical robustness was also tested. The method was applied to different type of samples: namely, pharmaceutical formulations, a photographic solution and irrigation and residual superficial waters.     

Thermo-optical "canard orbits" and excitable limit cycles

We demonstrate experimentally and theoretically the existence of canard orbits and excitable quasiharmonic limit cycles in the thermo-optical dynamics of semiconductor optical amplifiers. We also observe the phase locking of the noise-induced spikes to the small-amplitude Hopf quasiharmonic oscillations, recently predicted by Makarov, Nekorkin, and Velarde [Phys. Rev. Lett. 86, 3431 (2001)]].     

o-Dianisidine: a new reagent for selective spectrophotometric, flow injection determination of chlorine.

A flow injection analysis (FIA) procedure for the determination of free chlorine in industrial formulations and water samples is proposed. The manifold is provided with a gas-diffusion unit which permits the removal of interfering species and also the preconcentration of chlorine. The determination of chlorine is performed on the basis of the oxidation by o-dianisidine as a chromogenic reagent to a coloured product which can be monitored at 445 nm. The method (for a preconcentration step of 60 s) is linear over the range 0.04-1.00 mg l(-1) of chlorine, the limit of detection is 0.04 mg l(-1), the reproducibility of the procedure (as RSD of the slope) is 3.7% for a series of four independent calibrations, the precision (as RSD of a series of 30 continuous FIA peaks of 0.56 mg l(-1) of chlorine) is 1.4% and the sample throughput is 40 h(-1). A detailed comparative study of the analytical characteristics of a single mono-channel reverse FIA assembly and the same system but provided with a Fluoropore membrane filter of 0.5 microm pore size was performed to check the advantages of the new approach in terms of sensitivity, selectivity and limit of detection.