Supercritical fluid chromatography coupled to mass spectrometry: A valuable tool in food analysis

Producción CientíficaSupercritical fluid chromatography (SFC), although known for several decades, has undergone a growing interest in the last few years fueled by the introduction of modern instruments with improved robustness, and hyphenation to mass spectrometry (MS). This allows the analysis of trace compounds in complex samples with high selectivity, high sensitivity and in a short time, which has contributed to its increased use in the food analysis area. This work reviews the principal applications of SFC-MS in food analysis, highlighting the most important achievements

Development and validation of a new method for the simultaneous determination of spinetoram J and L in honey from different botanical origins employing solid-phase extraction with a polymeric sorbent and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Producción CientíficaThe objective of this study was to propose a novel method to determine residues of the bio-insecticide spinetoram, which is a mixture of two components (spinetoram J and L), in honey from multifloral, rosemary and heather botanical origins; liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was the technique employed. An efficient sample treatment (recoveries between 82% and 95%) involving a solid phase extraction with a polymeric sorbent has been recommended, and no matrix effect was observed. Chromatographic analysis (4 min) was performed in reverse phase mode by using a fused-core column (Kinetex® EVO C18) with acetonitrile and ammonium formate as the mobile phase components, which was applied in isocratic elution mode. Method was validated according to the current European legislation. Not only was it selective, but it also displayed a wide linear range, good precision (relative standard deviation values lower than 9%) and sensitivity (low limits of detection (spinetoram J, 0.1–0.3 μg/kg; spinetoram L, 0.1–0.2 μg/kg) and quantification (spinetoram J, 0.3–1.2 μg/kg; spinetoram L, 0.4–0.7 μg/kg). Several honey samples were analyzed with this method and no spinetoram residues were found above the limits of detection.Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2013-00042-C10-03 y 06

Study of different chiral columns for the enantiomeric separation of azoles using supercritical fluid chromatography

Producción CientíficaThe enantiomeric separation of antifungal compounds is an arduous task in pharmaceutical and biomedical fields due to the different properties that each diastereoisomer presents. The enantioseparation of a group of fungicides (sulconazole, bifonazole, triadimefon and triadimenol) using supercritical fluid chromatography was achieved in this work. For this goal, four different chiral columns based on polysaccharide derivatives, as well as the effect of different chromatographic parameters such as temperature, type and percentage of organic modifier (methanol, ethanol and isopropanol), were thoroughly investigated. The inversion of the elution order of enantiomers as a result of a change in the stationary phase or organic modifier was also evaluated by employing a circular dichroism detector. The best separation conditions, in terms of the enantioresolution and analysis time, were obtained with the Lux® Cellulose-2 column using isopropanol as the organic modifier

Trace analysis of flubendiamide in bee pollen using enhanced matrix removal-lipid sorbent clean-up and liquid chromatography-electrospray ionization mass spectrometry

Producción CientíficaIn this work, a new method has been proposed with the aim of determining flubendiamide, a recently commercialized insecticide, in bee pollen by using liquid chromatography coupled to electrospray ionization mass spectrometry. For this purpose, a novel sample treatment has been proposed that has proven efficient in terms of recovery (average analyte recoveries were between 90% and 102%) and absence of matrix effect, and one which is effective, fast and selective. This involved a solvent extraction using an acetonitrile and water mixture, and a clean-up stage where, in addition to freezing, an enhanced matrix removal-lipid sorbent was successfully used for the first time with this matrix and analyte. The chromatographic conditions were also optimized, by selecting a C18 based column (Gemini® C18) and acetic acid (1 mM) in water and methanol as mobile phase components, allowing elution of flubendiamide in<4 min, with a total analysis time of 14 min. Validation was carried out, with the result that all the parameters studied complied with existing European legislation. It should be noted that the sensitivity of the method was excellent, with a quantification limit (4 μg/kg) well below the maximum residue level established for this insecticide in bee products (50 μg/kg). Finally, several bee pollen samples were analyzed, and flubendiamide residues were not found in any of the cases.Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2013-00042-C10-03 y 06)

Estudio de la estabilidad de aptámeros generados frente a la proteína 4E-BP1

El control de la síntesis de proteínas juega un papel importante en el crecimiento y proliferación celular. En la mayoría de los organismos eucariotas se lleva a cabo una traducción dependiente de cap en la cual el factor eucariótico de iniciación (eIF) 4E juega un papel muy importante. La actividad de este factor está regulada por la proteína 4E-BP1 (proteína de unión a eIF4E), entre otras, cuya fosforilación permite la liberación del factor y su participación en la traducción. La sobreexpresión del factor eIF4E produce irregularidades en el ciclo celular relacionadas con el cáncer, enfermedad que produce 8.2 millones de muertes anuales en el mundo. La actividad reguladora de la proteína 4E-BP1 la convierte en una posible diana terapéutica, por lo que en el laboratorio se seleccionaron tres aptámeros frente a dicha proteína a través del método SELEX. Los aptámeros se conocen como 1R, 1F y 20F y poseen estructuras secundarias complejas y con posibilidad de formación de G-cuádruplex, lo que les confiere una alta estabilidad. Por ello en el presente trabajo se ha ampliado la caracterización estructural y funcional de estos aptámeros mediante parámetros como susceptibilidad a nucleasas, IC50, niveles intracelulares, efecto sobre la síntesis de proteínas y constante de disociación. Los resultados posicionan a los aptámeros 1R y 20F como los más estables y los principales candidatos como potenciales herramientas terapéuticas. Sin embargo, es necesario realizar nuevos experimentos funcionales así como estudiar la especificidad de los aptámeros frente a otras proteínas y otros aspectos importantes para su aplicación clínic

Differentiation of bee pollen samples according to their intact-glucosinolate content using canonical discriminant analysis

Producción CientíficaA study is presented of the real possibilities of glucosinolate content and chemometrics (canonical discriminant analysis) to differentiate bee pollen samples from four different apiaries (Fuentelahiguera, Monte, Pistacho, Tío Natalio) located in the same geographical area. Fifteen intact-glucosinolates were quantified by means of ultraperformance liquid chromatography coupled to a quadrupole time-of-flight mass detector in forty-nine bee pollen samples. Glucosinolate residues were detected in most of the samples, and these differed in number and concentration. It was possible to directly differentiate one of the apiaries (Fuentelahiguera) from the other three (Monte, Pistacho y Tío Natalio) by comparing glucosinolate content. These three apiaries were differentiated by means of the first two canonical variables obtained from a canonical discriminant analysis. Following this analysis, more than 88% of the samples could be assigned correctly to the Pistacho and Monte apiaries, and 100% to the Tío Natalio apiary.Este trabajo forma parte de los proyectos de investigación financiados por el Ministerio de Economía y Competitividad e INIA-FEDER (RTA2015-00013-C03-01 y 03)

Differentiation of bee pollen samples according to the apiary of origin and harvesting period based on their amino acid content

Producción CientíficaBee pollen is currently one of the most widely consumed dietary supplements due to its high nutritional value and its potentially beneficial effects on health. Unfortunately, in recent years an increase in the fraudulent marketing of this product has been detected, mainly in terms of adulteration with pollen from other sources. This has made it necessary to seek new tools to ensure its authentication. Therefore, this study investigates the use of free amino acids as markers of the geographical origin and harvesting period of bee pollen. To demonstrate their potential as biomarkers, 72 samples from four apiaries (Pistacho, Tío Natalio, Monte and Fuentelahiguera), located in the same geographical area (Marchamalo, Guadalajara, Spain), were analyzed by liquid chromatography-fluorescence detection, with the data obtained undergoing canonical discriminant analysis. Variable amounts and numbers of free amino acids were found in the samples analyzed; proline predominated in all of them, in a concentration range of 298–569989 mg/kg. The differences observed in amino acid composition could be attributed to the flowering plants from which the bee pollen samples originated. In addition, it was possible to statistically assign over 75% of the samples to the corresponding apiary of origin, the best results being obtained for the Fuentelahiguera and Tío Natalio apiaries (100%); this classification was even superior in the case of the harvesting periods, as more than 90% of the samples were correctly assigned, and in one period (June) a 100% rate was obtained.Ministerio de Economía, Industria y Competitividad (projects RTA 2015-00013-C03-01 and RTA 2015-00013-C03-03

Validez de los criterios DSM-IV según respuesta de los padres en el diagnóstico del trastorno por déficit de atención con hiperactividad.

Introducción. Objetivos: Estudiar la validez para el diagnóstico del trastorno por déficit de atención con hiperactividad (TDAH), de cada uno de los ítems DSM-IV y buscar un modelo reducido de ítems que ayude a detectar casos de niños con TDAH. Sujetos y método. Se utilizan los datos de un estudio epidemiológico sobre TDAH con una muestra de 1095 casos. El 6.6% son TDAH. Casos de TDAH definidos según ADHD RS-IV y criterios clínicos DSM-IV. Controles definidos por exclusión. Resultados. El modelo de regresión logística que mejor predice el fenotipo inatento está compuesto por los ítems del ADHD RS-IV (versión padres) 1, 3, 9, 15 y 17 (Se: 96.7%, Es: 81.5%); el fenotipo hiperactivo/impulsivo por los ítems 2, 4, 10, 12, 14 y 16 (Se: 96.6%, Es: 81%) y el fenotipo combinado por los ítems 9, 10, 12, 14 y 15 (Se: 100 %, Es: 82.6%). Existe una reducción del 66% de los ítems en el fenotipo combinado. Conclusiones. Es posible reducir la lista de síntomas de TDAH con unos niveles de validez adecuados y determinados ítems parecen tener mayor capacidad para determinar decisiones diagnóstica

Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain

The development of clinica lpractice recommendations or guidelines for the clinical use of biomarkers is an issue of great importance withr regard to adverse drug reactions.The poten-tial of pharmacogenomicbiomarkers has been extensively investigated in recent years.However,several barriers to implementing the use of pharmacogenomics testing exist.We conducted a survey among members of the Spanish Societies of Pharmacology and Clinical Pharmacology to obtain information about the perception of such barriers and to compare the perceptions of participants about the relative importance of majorgene/drug pairs.Of 11 potential barriers,the highest importance was attributed to lack of institutional support for pharmacogenomic stesting,and to the issues related to the lack of guidelines.Of the proposed gene/drug pairs the highest importance was assigned to HLA-B/abacavir, UGT1A1/irinotecan, and CYP2D6/tamoxifen.In this perspective article,we compare the relative importance of 29 gene/drugpairs in the Spanish study with that of the same pairs in the American Society for Clinical Pharmacology and Therapeutic sstudy,and we provide suggestions and areas of focus to develop a guide for clinical practice in pharmacogenomics testingThe work in the author’s laboratory is financed by Grants PS09/00943, PS09/00469, RETICS RIRAAF RD07/0064/0016, and CIBERehd from Instituto de Salud CarlosIII,Madrid, Spain, and by Grants GR10068 from Junta de Extremadura, Spain. Financed in part with FEDER funds from the European Unio