Determination of 5-nitroimidazoles and metabolites in environmental samples by micellar electrokinetic chromatography

Posted on 13 July, 2012 by fqm302

A method based on micellar electrokinetic chromatography (MEKC) with UV detection has been developed for the determination of nine 5-nitroimidazoles (5-NDZs), including metabolites in river water samples. Due to the relative insensitivity of UV detection in MEKC, a solid-phase extraction (SPE) method has been proposed that preconcentrates water samples fiftyfold and cleans them up off-line. An on-line preconcentration approach based on sweeping and the use of an extended light path fused-silica capillary (64.5 cm × 50 μm i.d., 56 cm effective length) was also found to improve the sensitivity of the method. Separation was carried out in <21 min using 20 mM phosphate buffer (pH 6.5) and 150 mM SDS as the background electrolyte (BGE). The temperature of the capillary was kept constant at 20°C, a voltage of 25 kV was applied (normal mode), and a detected wavelength of 320 nm was utilized. Hydrodynamic injection (50 mbar for 15 s) of the samples, which were dissolved in 20 mM phosphate (pH 6.5), was employed. The limits of detection were lower than 1.1 μg L−1. Recoveries of >80% from spiked river water samples were obtained for most of the analytes at three different concentration levels with acceptable precision. This method could provide an efficient and economical alternative to the use of chromatographic methods to monitor nitroimidazole residues, thus supplementing the relatively few methods available for the analysis of these compounds in environmental samples.

Posted in 5-nitroimidazoles, MEKC, SPE, Sweeping, UV-vis, Water | Tagged | Comments Off on Determination of 5-nitroimidazoles and metabolites in environmental samples by micellar electrokinetic chromatography

Convenient solid phase extraction of cephalosporins in milk using a molecularly imprinted polymer

Posted on 26 June, 2012 by fqm302

In this paper, a molecularly imprinted polymer (MIP) for cephalosporin molecules (cephalexin (CFL) and cephapirin (CFP)), was prepared by non covalent molecular imprinting approach and applied to solid phase extraction (SPE). For MIP synthesis, a tributylammonium cefadroxil salt (TBA–CFD) was used as template with methacrylic acid and ethylene glycol dimethacrylate as monomer and cross-linker, respectively, in acetone–methanol 92/8 (v/v) mixture. The selectivity of MIP versus non imprinted polymer (NIP) was confirmed for CFL, CFD and CFP in standard solutions as well as in milk samples. The efficiency of the synthesized MIP was evaluated by means of the application of the proposed MIP–SPE procedure to spiked milk samples previous to the HPLC method for the detection of cephalosporins. The MIP–SPE recoveries were higher than 60% for the three target analytes in spiked milk.

Posted in Cephalosporins, MIPs, Milk | Tagged | Comments Off on Convenient solid phase extraction of cephalosporins in milk using a molecularly imprinted polymer

Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction

Posted on 26 June, 2012 by fqm302

A method based on reverse phase capillary high performance liquid chromatography (capillary HPLC) coupled to laser-induced fluorescence detection (LIF) has been proposed for the determination of ochratoxin A (OTA) in wine samples. An anionic micellar medium was added to the mobile phase for increasing the fluorescence intensity and peak efficiency. Dispersive liquid–liquid microextraction (DLLME) has been used as a simple and efficient sample pretreatment method for the analysis of OTA in wines, being optimised by means of experimental design. The limit of detection was 5.5 ng L−1 (3 × S/N) and recoveries for different wines ranged from 91.7 to 98.1%. The proposed methodology could be classified as a green analytical chemistry alternative, combining the low organic solvent volumes required in the DLLME with the reduced consumption of mobile phase in capillary HPLC. The use of LIF as detector provided an extremely sensitive method for the determination of OTA in wines.

Posted in Capillary HPLC, DLLME, LIF, Mycotoxins, Wine | Tagged | Comments Off on Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction

Dispersive liquid-liquid microextraction using a low density extraction solvent for the determination of 17 N-methylcarbamates by micellar electrokinetic chromatography-electrospray-mass spectrometry employing a volatile surfactant

Posted on 23 June, 2012 by fqm302

A new analytical method based on micellar electrokinetic chromatography tandem mass spectrometry (MEKC–ESI–MS/MS) employing a MS friendly surfactant (ammonium perfluorooctanoate) is proposed and validated for the identification and simultaneous quantification of 17 N-methylcarbamate pesticides in environmental and drinking water samples. MS/MS detection using an ion trap as analyzer operating in the multiple reaction monitoring mode was used. Different parameters were optimized in order to obtain an adequate CE separation combined with the highest sensitivity in MS/MS. Dispersive liquid–liquid microextraction (DLLME) using a low-density extraction solvent has been proposed for extraction, obtaining a preconcentration factor of 10. Under optimum conditions, recoveries for fortified samples ranged from 83% to 101%, with relative standard deviations lower than 8%. The limits of detection ranged from 1 to 144 ng l−1, demonstrating the sensitivity and applicability of this fast, simple, and environmentally friendly method.

Posted in Carbamates, DLLME, MEKC, Mass spectrometry, Water | Tagged | Comments Off on Dispersive liquid-liquid microextraction using a low density extraction solvent for the determination of 17 N-methylcarbamates by micellar electrokinetic chromatography-electrospray-mass spectrometry employing a volatile surfactant

Determination of quinolones of veterinary use in bee products by ultra-high performance liquid chromatography–tandem mass spectrometry using a QuEChERS extraction procedure

Posted on 28 February, 2012 by fqm302

A reliable and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been developed for the determination of the eight quinolones of veterinary use regulated by European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, flumequine and oxolinic acid). Chromatographic conditions were optimized in order to increase sample throughput and sensitivity. The antibiotics were detected by electrospray ionization in positive ion mode with multiple reaction monitoring (MRM) and MS/MS conditions were optimized in order to increase selectivity, selecting the corresponding product ions for quantification and identification. The separation was achieved in 3 min, using a Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm), with a mobile phase of 0.02% aqueous formic acid solution and acetonitrile. A dispersive solid phase extraction methodology, often referred to as the “QuEChERS” (quick, easy, cheap, effective, rugged, and safe) method, was optimized for extraction of the quinolones from honey and also it was evaluated for other bee products such as royal jelly and propolis. The method was validated for each matrix in terms of linearity, trueness, precision, limits of detection (LODs) and quantification (LOQ). LODs ranged between 0.2 and 4.1 μg/kg with precision lower than 12% and satisfactory recoveries in most cases. The method was also applied for studying the occurrence of these antibiotics in several market samples.

Posted in Honey, Mass spectrometry, Propolis, QuEChERS, Quinolones, Royal jelly, UHPLC | Comments Off on Determination of quinolones of veterinary use in bee products by ultra-high performance liquid chromatography–tandem mass spectrometry using a QuEChERS extraction procedure

Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest

Posted on 21 December, 2011 by fqm302

We review analytical methodologies using capillary electrophoresis and related  techniques (micellar electrokinetic chromatography and capillary electrochromatography) with detection systems (ultraviolet-visible spectrometry, fluorescence, laser-induced fluorescence and mass spectrometry) for quantification of drugs of abuse and their metabolites in biological specimens of interest in forensic toxicology (e.g., blood, urine and hair). Despite some drawbacks that still need to be addressed and finally overcome when using this technique in forensic laboratories, the coupling of capillary electrophoresis and mass spectrometry generally provides a powerful option for detection and determination of very low concentrations of these compounds in some forensic matrices (e.g., hair).

Posted in Drugs, Electrophoresis, Review | Comments Off on Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest

Determination of carbamates at trace levels in water and cucumber by capillary liquid chromatography

Posted on 23 November, 2011 by fqm302

A sensitive and reliable method using capillary HPLC with UV-diode array detection (DAD) has been developed and validated for trace determination of carbamate pesticides in cucumber and environmental water samples. The analytes, including carbofuran, carbaryl, methiocarb, promecarb, benthiocarb and fenoxycarb, present legal residue levels regulated by the EU Council Directive 98/83/EC on drinking water and by the Regulation (EC) No. 396/2005 on vegetables. A previous off-line solid-phase extraction (SPE) procedure was required for preconcentration and sample clean-up. The separation was achieved using a C18 column (150 mm × 0.5 mm I.D, 5 µm particle size) and a mobile phase consisting of ACN : water using gradient mode, with a flow rate of 10 µl min−1. Taking advantages of the characteristics of capillary HPLC, low volume of sample and solvents were required, achieving limits of detection for the studied compounds ranged from 10.0–29.6 ng l−1 for water samples and 1.8–5.6 µg kg−1 for cucumber, using UV-detection. Recoveries studies for fortified samples, at three different concentration levels, were carried out obtaining recoveries ranging from 70.0 to 111.1% and relative standard deviations (RSDs) lower than 10.6%.

Posted in Capillary HPLC, Carbamates, Chromatography, Food, Pesticides, SPE, UV-vis, Vegetables, Water | Comments Off on Determination of carbamates at trace levels in water and cucumber by capillary liquid chromatography

Comparison of different sample treatments for the analysis of ochratoxin A in wine by capillary HPLC with laser-induced fluorescence detection

Posted on 2 November, 2011 by fqm302

Ochratoxin A (OTA) is a mycotoxin naturally found in various foods, including wine. As OTA is considered as a possible human carcinogen, the maximum concentration for this compound has been established at 2 μg kg−1 in wine by the EU (Directive (CE) No 1881/2006). Typically, immunoaffinity columns have been used for its extraction. However, simpler, more efficient and less contaminant extraction systems are demanding. In this work, dispersive liquid–liquid microextraction using ionic liquid as extractant solvent (IL-DLLME) and the QuEChERS procedure, have been evaluated and compared for extraction of OTA in wine samples. Laser-induced fluorescence (LIF, He–Cd Laser excitation at 325 nm) coupled with capillary HPLC has been used for the determination of OTA, using a sodium dodecyl sulfate micellar solution in the mobile phase to increase the fluorescence intensity. Matrix-matched calibration curves were established for both methods, obtaining LODs (3× S/N) of 5.2 ng·L−1 and 85.7 ng·L−1 for IL-DLLME and QuEChERS, respectively. Clean extracts were obtained for white, rose and red wines with both methods, with recoveries between 88.7–94.2% for IL-DLLME and between 82.6–86.2% for QuEChERS. The precision was evaluated in terms of repeatability (n = 9) and intermediate precision (n = 15), being ≤ 8.5% for IL-DLLME and ≤ 5.4% for QuEChERS.

Posted in Capillary HPLC, DLLME, Ionic liquids, LIF, Mycotoxins, QuEChERS, Wine | Tagged | Comments Off on Comparison of different sample treatments for the analysis of ochratoxin A in wine by capillary HPLC with laser-induced fluorescence detection

Desarrollo de un método MEKC para la determinación de 5-nitroimidazoles en aguas de río

Posted on 20 September, 2011 by fqm302

Los 5-nitroimidazoles (5-NDZs) son antimicrobianos con propiedades antibacterianas y antiprotozoarias. Debido a estas características se ha extendido su uso tanto como antibióticos humanos como veterinarios, mostrando eficacia contra la mayoría de bacterias anaerobias Gram negativas y algunas Gram positivas (Bacteroides grupo fragilis, Fusobacterium spp., Clostridium spp., Veilonella spp., incluyendo Clostridium difficile y C. perfringens), así como contra protozoos tales como Trichomonas vaginalis, Entamoeba hitolytica, Guardia lamblia y Balantidium coli.

Existen informes que atribuyen propiedades genotóxicas, mutagénicas y carcinogénicas a los 5-NDZs. Además, se trata de compuestos de alta solubilidad en el agua y baja biodegradabilidad, lo que favorece su bioacumulación. Estos problemas, además de la aparición de resistencias bacterianas a los 5-NDZs, han llevado a la Unión Europea (UE) a prohibir la utilización de estas sustancias como antibióticos veterinarios.

Pese a esta prohibición, la Agencia de Seguridad Alimentaria Europea (EFSA) sigue registrando aletas sobre alimentos de origen animal que contienen trazas de 5-NDZs. Esto supone que actualmente se está incumpliendo el Reglamento (UE) Nº37/2010, el cual establece que cualquier alimento de origen animal destinado al consumo humano debe estar exento de este tipo de antibióticos.

Como medida de control, y con objeto de garantizar el cumplimiento de la legislación vigente, se requieren de métodos analíticos que permitan la detección y cuantificación de los 5-NDZs, tanto en muestras de origen alimentario como medio ambiental. Por este motivo se ha desarrollado, dentro del Grupo de Investigación “Química Analítica Alimentaria, Ambiental y Clínica (FQM-302)”, un método analítico para la determinación de los mencionados antibióticos., el cual es descrito en la presente Memoria.

El método desarrollado emplea la Cromatografía Capilar Electrocinética Micelar, una modalidad de Electroforesis Capilar que ha permitido la cuantificación del metronidazol (MNZ), metronidazol-OH (MNZ-OH), dimetridazol (DMZ), ipronidazol (IPZ), dimetridazol-OH (HMMNI), ipronidazol-OH (IPZ-OH), ronidazol (RNZ), ornidazol (ORZ) y ternidazol (TRZ). Dicho método se ha aplicado a la determinación de residuos de estos compuestos en muestras de agua del río Genil (Granada).

Posted in 5-nitroimidazoles, Drugs, MEKC, SPE, Trabajo fin máster, UV-vis, Water | Comments Off on Desarrollo de un método MEKC para la determinación de 5-nitroimidazoles en aguas de río

Evaluación de la microextracción líquido-líquido dispersiva para la determinación de patulina en zumos de manzana mediante electroforesis capilar

Posted on 20 September, 2011 by fqm302

La contaminación microbiológica de los alimentos es la causa principal de las enfermedades de origen alimentario. Dentro de este tipo de contaminación destacan las micotoxinas, sustancias producidas por ciertos hongos pertenecientes principalmente a los
géneros Aspergillus, Fusarium y Penicillium. Estas sustancias suelen encontrarse en una gran variedad de productos agrícolas, almacenados en condiciones de humedad y aerobiosis, y son los contaminantes naturales de los alimentos más extendidos a nivel mundial. Se trata de productos altamente tóxicos, los cuales pueden producir mutaciones, cáncer, malformaciones en los fetos y disminuir la actividad del sistema inmune. Debido a su gran variedad de efectos tóxicos y, sobre todo, a su extrema resistencia al calor (termorresistencia), la presencia de las micotoxinas en los alimentos es considerada de alto riesgo para la salud humana y de los animales. La contaminación de los alimentos con micotoxinas es dependiente de las condiciones ambientales, que pueden propiciar el crecimiento del hongo y, por tanto, la producción de las toxinas. De este modo, la mayoría de los productos agrícolas pueden ser susceptibles de contaminación casi en cualquier momento, desde su producción en el campo, durante la cosecha, en el transporte y en el almacenamiento.

La patulina es una micotoxina producida por los hongos Penicillium griseofulvum (P. patulum o P. urticae) en cereales y nueces, P. expansum en manzanas y en gran variedad de frutas, P. gladioli y P. sclerotigenum en rizomas y bulbos. Estos hongos contaminan principalmente en la etapa de postcosecha de la fruta, ya que la mayoría de las especies productoras de patulina crecen a temperaturas de refrigeración. La patulina, al igual que las demás micotoxinas, puede ser neurotóxica, inmunotóxica, inmunosupresiva, genotóxica, teratogénica y carcinogénica. Como resultado, la legislación a nivel europeo sobre seguridad alimentaria es cada vez más restrictiva en cuanto a los niveles de patulina en alimentos y ha establecido los contenidos máximos de esta micotoxina mediante el Reglamento (CE) N º 1881/2006 [1]. Estos contenidos máximos se han establecido en 50 μg/kg para zumos de frutas, zumos de frutas concentrados reconstituidos y néctares de frutas, en 25 μg/kg para productos sólidos elaborados con manzanas, incluidos la compota y el puré de manzana destinados al consumo directo y, por último, en 10 μg/kg para zumo de manzana y productos sólidos elaborados a base de manzanas, incluidos la compota y el puré de manzana, destinados a los lactantes y niños de corta edad y para alimentos infantiles distintos de los alimentos elaborados a base de cereales lactantes y niños de corta edad.

Los métodos desarrollados para la determinación de patulina, habrán de presentar, por tanto, una adecuada selectividad, permitiendo efectuar análisis sin que interfieran los componentes de la matriz, así como una alta sensibilidad. De este modo, la etapa de tratamiento de muestra será, por tanto, decisiva en el diseño de tales métodos.

En los últimos años existe un creciente interés en la simplificación y miniaturización de los sistemas de tratamiento de muestra, introduciendo disolventes menos contaminantes y disminuyendo considerablemente los volúmenes utilizados, en línea con los principios de la llamada Química Verde o Química beneficiosa para el medio ambiente. Se trata de diseñar
productos y procesos químicos que reduzcan o eliminen el uso y producción de sustancias peligrosas, implicando una mayor seguridad y un menor coste en relación a los procesos convencionales. Así, la microextracción líquido-líquido (liquid phase microextraction, LPME) es una técnica simple y económica en la que se requieren sólo unos microlitros de disolvente para concentrar a los analitos a partir de las muestras, en lugar de los elevados volúmenes requeridos en la extracción líquido-líquido convencional.

Este trabajo tiene como objeto principal la puesta a punto de una metodología de tratamiento de muestra LPME recientemente desarrollada denominada microextracción líquido-líquido dispersiva (dispersive liquid phase microextraction, DLLME), previa al análisis de patulina en zumos de manzana, que presenta la ventaja de ser eficaz, rápida y sencilla, además de reducir el consumo de disolventes orgánicos. Para ello, se hará uso de la cromatografía capilar electrocinética micelar (micellar electrokinetic capillary chromatography, MEKC) con detección UV-VIS, como técnica de separación, dada la naturaleza de la molécula y las ventajas que presenta esta metodología frente a los métodos habituales basados en cromatografía líquida.

Posted in DLLME, Juice, MEKC, Mycotoxins, Trabajo fin máster, UV-vis | Tagged | Comments Off on Evaluación de la microextracción líquido-líquido dispersiva para la determinación de patulina en zumos de manzana mediante electroforesis capilar