Olive oil quantification of edible vegetable oil blends using triacylglycerols chromatographic fingerprints and chemometric tools.

The present work studies the effectiveness of the use of triacylglycerols (TAGs) for the quantification of olive oil in blends with vegetable oils. The determinations were obtained using high-performance liquid chromatography (HPLC) coupled to a Charged Aerosol Detector (CAD), in combination with Partial Least Squares (PLS) regression and using interval PLS (iPLS) for variable selection. Results revealed that PLS models can predict olive oil concentrations with reasonable errors. Variable selection through iPLS did not improve predictions significantly, but revealed the chemical information important in the chromatogram to quantify olive oil in vegetable oil blends.

Discriminating olive and non-olive oils using HPLC-CAD and chemometrics.

This work presents a method for an efficient differentiation of olive oil and several types of vegetable oils using chemometric tools. Triacylglycerides (TAGs) profiles of 126 samples of different categories and varieties of olive oils, and types of edible oils, including corn, sunflower, peanut, soybean, rapeseed, canola, seed, sesame, grape seed, and some mixed oils, have been analyzed. High-performance liquid chromatography coupled to a charged aerosol detector was used to characterize TAGs. The complete chromatograms were evaluated by PCA, PLS-DA, and MCR in combination with suitable preprocessing. The chromatographic data show two clusters; one for olive oil samples and another for the non-olive oils. Commercial oil blends are located between the groups, depending on the concentration of olive oil in the sample. As a result, a good classification among olive oils and non-olive oils and a chemical justification of such classification was achieved.

[Epidemiology and costs of severe sepsis in Madrid. A hospital discharge study]. / Epidemiología y costes de la sepsis grave en Madrid. Estudio de altas hospitalarias.

BACKGROUND AND OBJECTIVE: Severe sepsis is a complex syndrome to define, diagnose and treat. This population-based study describes the epidemiology of sepsis in the Region of Madrid, estimates its incidence and mortality, and assesses its impact on hospital stays and costs. PATIENTS AND METHODS: The source of information was the Minimum Basic Hospital Data Set from the Region of Madrid in 2001. Severe sepsis cases were defined as discharges with a combination of organic failure and presence or suspicion of infection through a combination of codes previously proposed and utilized. A descriptive study was performed, incidence rates were calculated, lengths of stay and costs were estimated, and mortality was analyzed. RESULTS: 6,968 episodes were identified. Mean age was 62.5 year. 59.7% were male. Annual incidence was 14.1/10,000 inhabitants, being highest for those 84 and older (230.8/10,000). 1.7 infections per episode were detected. More frequently identified microorganisms were Streptococcus sp., Staphylococcus sp., Escherichia coli and Candida sp. The most frequent organic dysfunctions were renal (39.7%) and respiratory (35.7%). Mortality was 33%. Mortality was higher in cases with more than one organic failure, hepatic dysfunction or cancer. Mean length of stay was 28.9 day. Annual overall costs were 70 million euros. CONCLUSIONS: Severe sepsis is a frequent process, with a high mortality and a significant impact on health care resource utilization.

Epidemiología y costes de la sepsis grave en Madrid. Estudio de altas hospitalarias / Epidemiology and costs of severe sepsis in Madrid. A hospital discharge study

Fundamento y objetivo. La sepsis grave es un síndrome complejo de definir, diagnosticar y tratar. Este trabajo de base poblacional describe la epidemiología de la sepsis grave en la Comunidad de Madrid, estima su incidencia y mortalidad y evalúa su impacto en estancias y costes. Pacientes y método. La fuente de información fue el conjunto mínimo básico de datos de la Comunidad de Madrid del año 2001. Se definieron como casos de sepsis grave aquellos en los que existía la presencia de fracaso orgánico y presencia o sospecha de infección a partir de la combinación de códigos de enfermedad y códigos de procedimientos utilizando criterios propuestos y utilizados previamente. Se efectuó un estudio descriptivo, se calcularon tasas poblacionales de incidencia de sepsis, se obtuvieron las estancias medias, se estimó el coste y se analizó la mortalidad por sepsis. Resultados. Se identificaron 6.968 episodios. La edad media fue de 62,5 años. El 59,7% eran hombres. La incidencia anual fue de 14,1/10.000 habitantes, siendo máxima en los mayores de 84 años (230,8/10.000). Se detectaron 1,7 infecciones por episodio. Los microorganismos más frecuentes fueron Streptococcus sp., Staphylococcus sp., Escherichia coli y Candida sp. Las disfunciones orgánicas más frecuentes fueron renal (39,7%) y respiratoria (35,7%). La mortalidad global fue de un 33% y era superior para los episodios con más de una disfunción orgánica, disfunción hepática, o neoplasia. La estancia media fue de 28,9 días. El coste anual de la atención a la sepsis grave en la Comunidad de Madrid es de 70 millones de euros. Conclusiones. La sepsis grave es un proceso frecuente, presenta una elevada mortalidad y tiene un importante impacto en consumo de recursos asistenciales

Background and objective. Severe sepsis is a complex syndrome to define, diagnose and treat. This population-based study describes the epidemiology of sepsis in the Region of Madrid, estimates its incidence and mortality, and assesses its impact on hospital stays and costs. Patients and methods. The source of information was the Minimum Basic Hospital Data Set from the Region of Madrid in 2001. Severe sepsis cases were defined as discharges with a combination of organic failure and presence or suspicion of infection through a combination of codes previously proposed and utilized. A descriptive study was performed, incidence rates were calculated, lengths of stay and costs were estimated, and mortality was analyzed. Results. 6,968 episodes were identified. Mean age was 62.5 year. 59.7% were male. Annual incidence was 14.1/10,000 inhabitants, being highest for those 84 and older (230.8/10,000). 1.7 infections per episode were detected. More frequently identified microorganisms were Streptococcus sp., Staphylococcus sp., Escherichia coli and Candida sp. The most frequent organic dysfunctions were renal (39.7%) and respiratory (35.7%). Mortality was 33%. Mortality was higher in cases with more than one organic failure, hepatic dysfunction or cancer. Mean length of stay was 28.9 day. Annual overall costs were 70 million euros. Conclusions. Severe sepsis is a frequent process, with a high mortality and a significant impact on health care resource utilization

Chemiluminescence determination of amikacin based on the inhibition of the luminol reaction catalyzed by copper.

A simple and sensitive method has been proposed for the amikacin sulphate determination. It is based on the inhibition of the chemiluminescence (CL) emission generated from the oxidation of luminol in alkaline medium by H2O2 catalyzed by Cu(II), due to the interaction caused by amikacin, which forms a robust complex with the catalyst. The optimization of the experimental and instrumental variables affecting this CL inhibition effect has been carried out using statistical models, based on the application of two-level full factorial and Box-Behnken designs. The performance characteristics of the proposed method have been established, showing that the method is efficient to determine amikacin sulphate in the linear range of 9.89-20 mg/L with a detection limit of 2.97 mg/L. It has been successfully applied to the amikacin sulphate determination in pharmaceutical formulations.

An overview of qualimetric strategies for optimisation and calibration in pharmaceutical analysis using flow injection techniques.

Flow Injection analysis represents an attractive tool because of its great advantages, such as versatility, speed, high sampling rate and wide applicability in the field of pharmaceutical analysis. However, due to the inherent characteristics of the technique, the choice of the best set of operational and chemical conditions is complicated and the conventional univariate optimisation method present some limitations, mainly due to fact that the interdependence of variables is not considered. In relation to the calibration process, because of the transient character of the signals obtained using FIA manifolds coupled with different detection techniques, different strategies can be used in calibration to solve some problems related to the nature of the signal thereby improving the performance characteristics of the method. This paper offers an overview of different methodologies used in optimisation based on the use of statistically designed experiments and some strategies developed for calibration applied to the analysis of pharmaceuticals.

Applying non-parametric statistical methods to the classical measurements of inclusion complex binding constants.

A study on using non-parametric statistical methods was carried out to calculate the binding constant of an inclusion complex and to estimate its associated uncertainty. First, a correct evaluation of the stoichiometry was carried out in order to ensure an accurate determination of the binding constant. For this purpose, the modified Benesi-Hildelbrand method had been previously applied. Then, four statistical methods (three non-parametric methods: two bootstrap approaches, the jackknife method and a parametric one: Fieller's theorem) were employed in order to compute the binding constant. The results obtained from applying these methods and the combination of the methods: jackknife after bootstrap and bootstrap after jackknife were compared. The best results in terms of accuracy were obtained from the application of a bootstrap method: the resampling residuals approach. These procedures were applied to the inclusion complex 2-hydroxil-propyl-beta-cyclodextrin-2,4-dichloro-phenoxyacetic, which shows photochemically-induced fluorescence.

Use of factorial design and Doehlert matrix for multivariate optimisation of an on-line preconcentration system for lead determination by flame atomic absorption spectrometry.

A system for on-line preconcentration and determination of lead by flame atomic absorption spectrometry (FAAS) was proposed. It was based on the sorption of lead(II) ions on a minicolumn of polyurethane foam loaded with 2-(2-thiazolylazo)-5-dimethylaminophenol (TAM). The optimisation step was carried out using two-level full factorial and Doehlert designs for the determination of the optimum conditions for lead preconcentration. The proposed procedure allowed the determination of lead with a detection limit of 2.2 microg L(-1), and a precision, calculated as relative standard deviation (RSD), of 2.4 and 6.8 for a lead concentration of 50.0 and 10.0 microg L(-1), respectively. A preconcentration factor of 45 and a sampling frequency of 27 samples per hour were obtained. The recovery achieved for lead determination in the presence of several cations demonstrated that this procedure has enough selectivity for analysis of environmental samples. The validation was carried out by analysis of certified reference material. This procedure was applied to lead determination in natural food.

Monitoring of chemical and enzymatic hydrolysis of water-soluble proteins using flow-injection analysis with fluorescence detection and an aqueous eluant containing 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe.

The exposed hydrophobicity of proteins, which is due to the hydrophobic regions located on their surfaces, enhances the fluorescence intensity of the probe 2-p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) by the formation of a complex. During the hydrolysis of a protein, the average exposed hydrophobicity of the substrate continuously changes with incubation time, and these changes are immediately reflected by a corresponding change in the fluorescence intensity of the 2,6-TNS/substrate complex. Therefore, 2,6-TNS seems to be a good probe to monitor the course of the depolymerization processes of proteins. In this work, bovine serum albumin and alpha-casein have been hydrolyzed both chemically and enzymatically, and the course of the reactions is monitored by using flow-injection analysis (FIA) with fluorescence detection and a buffered aqueous eluant containing 2,6-TNS as the fluorescent probe. Results indicate that the time evolution of the fluorescence intensity of the 2,6-TNS/substrate complex can be correlated with the initial concentration of the parent protein, in mass per unit volume, the hydrolytic activity added, and the time evolution of the mean chain length of the substrate. In addition, because the time elapsed between injection of the sample into the FIA system and measurement of the corresponding fluorescence intensity is only a few seconds, this methodology could be a useful tool for on-line monitoring of processes for the production of protein hydrolysates.

Optimizing analytical methods using sequential response surface methodology. Application to the pararosaniline determination of formaldehyde.

Sequential response surface methodology is a general procedure to re-optimize common analytical methods on the basis of the application of the response surface methodology and of a new approach to the steepest ascent method. This procedure, which is easy to apply, consists of estimating an analytical function relating the response with the experimental parameters by means of a second-degree polynomial. Thus, a 2nd order design covering the total experimental domain is used and when a maximum is obtained, the characteristics of the response surface are confirmed using a new design, which is obtained contracting the first one. In the proposed methodology, Box-Behnken designs are used because they offer advantages in comparison with second order designs more frequently used in the steepest ascent method (central composite designs), i.e. fewer experiments are needed, they are more efficient, they can be moved through the experimental domain and they can even be easily contracted or expanded.

Micellar-enhanced photochemically induced fluorescence detection of chlorophenoxyacid herbicides. Flow injection analysis of mecoprop and 2,4-dichlorophenoxyacetic acid.

In this paper, a combination of a flow injection analysis (FIA) system with micellar-enhanced photochemically induced fluorescence (MEPIF) detection is presented as a powerful alternative for the rapid and sensitive analysis of chlorophenoxyacid herbicides. These compounds do not show native fluorescence but they can be photolysed into strongly fluorescence photoproducts after direct irradiation with ultraviolet light. The use of a cationic surfactant such as cetyltrimethylammonium chloride (CTAC) provides a considerable enhancement of photochemically induced fluorescence intensity and the nature of the technique allows a possible and easy adaptation to a FIA system. In this sense, parameters related to the nature of the analytical signal (pH, irradiation times, surfactant concentration) and to the FIA manifold (injection volume, flow rate and reactor length) have been optimised. Linear calibration graphs, with three replicates for each concentration value were established in the range of 0.2-5.0 mug ml(-1) for 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.1-5.0 mug ml(-1) for Mecoprop (MCPP). The IUPAC detection limits were 73.2 and 33.5 ng ml(-1) for 2,4-D and MCPP, respectively. Satisfactory recoveries were obtained in the analysis of these herbicides in spiked waters.

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system.

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates.

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.

A theoretical equation describing the time evolution of the concentration of a selected range of substrate molecular weights in depolymerization processes mediated by single-attack mechanism endo-enzymes.

Monitoring the time evolution of the concentration of a selected range of molecular weights of substrate, referred to as "detectable" substrate, has been used to determine endo-enzymic activities in polysaccharide depolymerizing processes. In the methodologies based on the use of dye-labeled substrates, the "detectable" substrate extends from a given molecular weight threshold downward. On the contrary, in the fluorescent probe-flow injection analysis methodology, initially developed to determine (1 --> 3)-(1 --> 4)-beta-D-glucanase activities, the "detectable" substrate extends from a given molecular weight threshold upward. Assuming that the time evolution of the molecular weight distribution of the substrate follows the most probable distribution (the enzymic attack is random and its mechanism is single attack), a theoretical equation describing the time evolution of the concentration of "detectable" substrate (from a given molecular weight threshold upward or downward) has been deduced. This equation, Wd = Wo. (1 + alphat). e-alphat, where Wd is the concentration of "detectable" substrate, Wo is the initial concentration of the substrate, t is the depolymerization time, and alpha is a parameter correlated through a hyperbola with the initial concentrations of enzyme and substrate and the Michaelis-Menten constant, Km, has been tested against different (1 --> 3)-(1 --> 4)-beta-D-glucan/(1 --> 3)-(1 --> 4)-beta-D-glucanase systems using the fluorescent probe-flow injection analysis methodology and Calcofluor as the fluorescent probe. The most important predictions of the theoretical equation, which allow accurate determination of both endo-enzymic activities and kinetic constants, have been experimentally confirmed.

Determination of proteins in the presence of imidazole buffers.

Imidazole buffers were found to interfere with the determination of soluble proteins using Lowry's classical method. The influence of the constituent elements of the buffer on the calibration line was studied statistically. By combining the data corresponding to different experimental sequences, interserial calibration curves for different concentrations of imidazole buffer (10-30 mM) were obtained. The absorbance-buffer volume dependence curves produced a good fit to second-order polynomials. The accuracy of protein determination in a medium with imidazole buffer, using appropriate calibration curves, were tested by comparison with the technique of multiple standard addition and by means of recovery studies. These experiments were performed on chick brain homogenate samples. Other important aspects of validation, such as sensitivity and accuracy, were also studied.

Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis.

Two polypeptides showing alpha-L-arabinofuranosidase activity have been purified to homogeneity from culture supernatants of a Bacillus subtilis clone harbouring the xynD gene [Gosalbes et al. (1991) J Bacteriol 173: 7705-7710] from Bacillus polymyxa. Both polypeptides, with determined molecular masses of 64 kDa and 53 kDa, share the same sequence at their N termini, which also coincides with the sequence deduced for the mature protein from the previously determined sequence of nucleotides (Gosalbes et al. 1991). The two polypeptides have been biochemically characterized. Arabinose is the unique product released from arabinose-containing xylans which are substrates for both enzyme forms. Other natural arabinose-containing polysaccharides, such as arabinogalactans, are not attacked by them but some artificial arabinose derivatives are good substrates for both polypeptides. Their arabinose-releasing activity on arabinoxylans facilitates the hydrolysis of the xylan backbone by some endoxylanases from Bacillus polymyxa.

Determination of vanadium by solid-phase spectrophotometry after its preconcentration as an Eriochrome Cyanine R complex on a dextran-type exchanger.

A method is described for the determination by solid phase spectrophotometry (SPS) of trace amounts of vanadium in natural water and crude petroleum samples. The procedure is based on fixation on a dextran-type anion exchanger of the complex V(IV)-Eriochrome Cyanine R. The absorbance of the gel, at 563 and 750 nm, packed in a 1 mm cell, is measured directly. Vanadium can be determined in the 0.6-25.0 mug l(-1) range with a relative standard deviation of 2.2%. The comparison of the SPS method and the gallic acid persulphate method shows that the linearity, analytical sensitivity and precision were better for the SPS method, and that the latter method has lower detection and quantification limits than the gallic acid persulphate method.

Purification and Characterization of Alkaline Xylanases from Bacillus polymyxa.

By applying different classical and fast protein liquid chromatographic techniques, three xylanases (beta-1,4-d-xylan xylanhydrolase) were purified to homogeneity from the extracellular enzymatic complex of Bacillus polymyxa. The three enzymes (X(34)C, X(34)E, and X(22)) were small proteins of 34, 34, and 22 kDa and basic pIs 9.3, >9.3, and 9.0, respectively. X(34)C and X(34)E are closely related and seem to be isoforms of the same enzyme. However, they differ in some characteristics. The three enzymes had different pH and temperature optima. One of them, X(34)E, showed a high thermal stability. The V(max) values determined for X(34)C, X(34)E, and X(22) enzymes on oat spelts xylan were 14.9, 85.5, and 64.0 U mg, respectively, and 16.1, 62.0, and 150.6 U mg on birchwood xylan. When oat spelts xylan was the substrate used, K(m) values of 3.4, 2.4, and 1.9 mg ml were obtained for X(34)C, X(34)E, and X(22) enzymes, respectively, and 0.65, 6.3, and 0.32 mg ml were the respective K(m) values determined with birchwood xylan as the substrate. The enzymes were nondebranching endo-beta-xylanases. Xylose was one of the products of xylan hydrolysis by xylanases X(34)C and X(34)E, but this monosaccharide was not released by X(22) enzyme. However, neither of the enzymes was able to degrade xylobiose.

[Evolution of mortality from principal chronic diseases in Spain 1975-1988]. / Evolución de la mortalidad por las principales enfermedades crónicas en España, 1975-1988.

BACKGROUND: Most mortality in developed countries is attributable to chronic non transmittable diseases, many of which are theoretically susceptible to prevention. The tendency of mortality by the principal chronic diseases in Spain is reviewed with different prevention strategies of the same being discussed. METHODS: The 9 chronic diseases which presented the highest mortality rate in Spain in 1988 are included. The rates of mortality, adjusted by age/year in males and females was calculated from the data of deaths by age, sex and cause of death from death statistics. Moreover, the percentage of the mean annual change of these during the periods 1975-1981 and 1982-1988 have also been calculated. RESULTS: Except for mortality by malignant tumor of the colon and rectum, malignant lung tumor in males and malignant breast tumors in women, which had an increase, the remaining diseases in the adjusted mortality rate by age decreased between 1975-1988. CONCLUSIONS: Among the diseases in which the rate of mortality has increased there is only that of malignant lung tumors for which one factor has consistently been identified as responsible for this increase, that being smoking. The possible influence of the control of arterial hypertension in the decrease in mortality of cerebrovascular disease must be emphasized. Moreover, the impact which the ninth review of the International Disease Classification had in the reduction in mortality by chronic bronchitis, emphysema and asthma must also be pointed out.