Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of carbamates in wines by ultra-high performance liquid chromatography–tandem mass spectrometry

Posted on 14 October, 2013 by fqm302

A new sensitive multiresidue method based on ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) has been developed for the detection, confirmation and quantification of twenty five carbamates in wine samples. The separation was achieved in 5.5 min, using a Zorbax Eclipse plus RRHD C18 column (50 mm × 2.1 mm, 1.8 μm), with a mobile phase of water and methanol, both of them with 0.01% formic acid. The analytes were detected in positive mode with multiple reaction monitoring mode. Ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME), using a low-density extraction solvent has been optimized for the satisfactory extraction of carbamates and clean-up of extracts. The matrix effect was studied, showing that the proposed procedure provides very clean extracts. Under optimum conditions, recoveries for fortified wine samples ranged from 74 to 102%, with relative standard deviations lower than 6%. Limits of quantification ranged from 0.15 to 0.92 μg l−1, showing the high sensitivity of this fast and simple method and its compliance with current requirements.

Posted in Carbamates, Mass spectrometry, UASEME, UHPLC, Wine | Tagged , | Comments Off on Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of carbamates in wines by ultra-high performance liquid chromatography–tandem mass spectrometry

Green methodology based on dispersive liquid–liquid microextraction and micellar electrokinetic chromatography for 5-nitroimidazole analysis in water samples

Posted on 19 September, 2013 by fqm302
Dispersive liquid–liquid microextraction has been proposed as an extraction technique combined with micellar  electrokinetic chromatography (MEKC) for the analysis of eight 5-nitroimidazole compounds, including some metabolites, in water samples. Determination has been carried out using a diode array detector, employing 20 mM sodium phosphate and150 mM SDS as separation buffer. Separation has taken place under a voltage of 25 kV anda temperature of 20 C. Samples were prepared in a buffer without micelles and they were hydrodynamically injected at 50 mbar for 25 s, producing a sweeping effect on the analytes for increasing sensitivity. Different factors involved in the dispersive liquid–liquid microextraction procedure were optimized, such as sample pH, nature, and volume of extraction and dispersive solvents in the mixture, percentage of NaCl added to sample and shaking time after the injection of the extraction and dispersive solvents. The method was characterized for water samples, achieving detection limits lower than 2.4g/L. Trueness was checked in river, tap, and bottled water. Dispersive liquid–liquid microextraction combined with MEKC constitutes an easy, cheap, and green alternative for 5-nitroimidazole analysis in environmental water samples.
Posted in 5-nitroimidazoles, DLLME, MEKC, Sweeping, UV-vis, Water | Tagged , | Comments Off on Green methodology based on dispersive liquid–liquid microextraction and micellar electrokinetic chromatography for 5-nitroimidazole analysis in water samples

Micellar electrokinetic chromatography–electrospray ionization mass spectrometry employing a volatile surfactant for the analysis of amino acids in human urine

Posted on 12 September, 2013 by fqm302

A new MEKC-ESI-MS method for the analysis of amino acids (AAs) in human urine was developed employing ammonium perfluorooctanoate (APFO) as volatile surfactant. The influence of APFO on the MS signal of AAs was evaluated by infusion experiments, which showed that APFO hardly affects analyte responses and presents significantly less ion suppression than equal concentrations of ammonium acetate. In order to obtain efficient separation of AAs, MEKC parameters such as the pH and APFO concentration of the BGE, were optimized. Optimum AA resolution, including baseline separation of leucine and isoleucine, was obtained using 150 mM APFO (pH 9.0) as BGE, representing a considerable selectivity improvement over CE using 50 mM ammonium acetate (pH 9.0). Optimization of CE-MS parameters, such as sheath liquid composition and flow rate, and ESI and MS settings, led to LODs ranging from 9 to 26 ng/mL for the 20 tested AAs, which is highly favorable for an MEKC-MS method. Good linearity (r2 > 0.99) and repeatability were obtained for all AAs tested with RSD values of 3.0–6.7% for peak area and <1.5% for migration time. The applicability of the MEKC-MS method was demonstrated by the quantitative determination of AAs in urine employing only a 1:1 dilution with BGE as sample pretreatment. All AAs could selectively be detected and quantified obtaining relevant concentration values for normal human urine.

Posted in Amino acids, MEKC, Mass spectrometry, Urine | Tagged , | Comments Off on Micellar electrokinetic chromatography–electrospray ionization mass spectrometry employing a volatile surfactant for the analysis of amino acids in human urine

Mass spectrometric and contactless conductivity detection approaches in the determination of muscle relaxants by capillary electrophoresis

Posted on 12 September, 2013 by fqm302

Novel and rapid capillary electrophoresis-coupled tandem mass spectrometry (CE-MS/MS) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) methods have been developed for the separation and determination of three neuromuscular blocking agents: pancuronium, vecuronium, and rocuronium. In both cases, the separation was conducted in background electrolytes based on acidic acetate-ammonium buffers to avoid possible decomposition of the analytes that are known to be unstable in alkaline media. Baseline resolution of the analytes was achieved in the presence of modified γ-cyclodextrin by CE with C4D detection. The two detection techniques were compared with regard to analytical figures of merit including linear dynamic range, limit of detection, limit of quantification, precision, and accuracy. The calibration curves showed good linearity for both detection methods examined (characterized by r2 ≥ 0.9908). The LODs of the CE-MS/MS and the CE-C4D methods differed at least by two orders of magnitude considering all analytes. The differences in precision and accuracy of these methods were evaluated and discussed. The assays of pancuronium, vecuronium, and rocuronium in commercial injection solutions by CE-MS/MS and CE-C4D were performed and the results compared.

Posted in C4D, CZE, Cyclodextrins, Mass spectrometry, Muscle relaxants, Pharmaceutical formulations | Tagged , | Comments Off on Mass spectrometric and contactless conductivity detection approaches in the determination of muscle relaxants by capillary electrophoresis

Novel solid phase extraction method for the analysis of 5-nitroimidazoles and metabolites in milk samples by capillary electrophoresis

Posted on 9 September, 2013 by fqm302

A new sample treatment has been developed for the extraction of 5-nitroimidazole (5-NDZ) drugs in milk samples previous to their determination by micellar electrokinetic chromatography (MEKC). Fat removing and protein precipitation were simultaneously carried out by the addition of trichloroacetic acid (TCA) and subsequent centrifugation. Clean-up and off-line concentration were achieved by a novel solid-phase extraction (SPE) method employing mixed cation exchange (MCX) cartridges, obtaining an off-line concentration factor of 18. Analyses were performed in less than 18 min employing 20 mM phosphate buffer (pH 6.5) and 150 mM SDS as background electrolyte (BGE). During the separation procedure a temperature of 20 °C and a voltage of 25 kV (normal mode) were applied. Due to sweeping effects, an on-line concentration was achieved for all the studied compounds and detection limits lower than 1.8 μg L−1 were obtained. This method has been successfully applied to milk samples of different origins, including raw ewe milk.

Posted in 5-nitroimidazoles, MEKC, Milk, SPE, Sweeping, UV-vis | Tagged , | Comments Off on Novel solid phase extraction method for the analysis of 5-nitroimidazoles and metabolites in milk samples by capillary electrophoresis

On-line anion exchange solid-phase extraction coupled to liquid chromatography with fluorescence detection to determine quinolones in water and human urine

Posted on 5 September, 2013 by fqm302

An analytical method based on on-line solid-phase extraction coupled to liquid chromatography with fluorescence detection has been developed to determine quinolones in tap water and human urine. A home-made setup was used to percolate 10 mL of sample through a solid-phase extraction column. Analytes were retained onto the sorbent by an anion exchange mechanism which ensures an optimum compatibility with the subsequent chromatographic separation. A C-18 column containing core-shell particles (2.6 μm) was used to achieve peak efficiencies up to 200,000 plates/m, at a flow rate of 1.2 mL/min and without the need for special pumps. The method allowed the determination of 11 quinolones directly in tap water samples in less than 20 min and with limits of detection ranging between 7 and 110 ng/L. The sensitivity achieved made possible the direct determination of 9 quinolones in human urine without any sample treatment, just dilution with water. Relative recoveries between 94 and 109% were obtained meaning that the matrix effect in human urine is negligible after dilution. Satisfactory results were also obtained in terms of precision since relative standard deviations were always below 13%.

Posted in Fluorescence, Quinolones, Urine, Water, on-line SPE | Tagged , | Comments Off on On-line anion exchange solid-phase extraction coupled to liquid chromatography with fluorescence detection to determine quinolones in water and human urine

Simple methodology for the determination of mycotoxins in pseudocereals, spelt and rice

Posted on 4 September, 2013 by fqm302

Nowadays the interest and consumption of pseudocereals is increasing due to their nutritional properties. Like cereals and oilseeds, pseudocereal seeds are susceptible to fungal growth and mycotoxin contamination; however these matrices have received little attention in literature. A sensitive, simple and rapid method for the determination of fifteen mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, fumonisin B1, fumonisin B2, nivalenol, deoxynivalenol, fusarenon-X, T-2 and HT-2 toxin, citrinin, sterigmatocystin and zearalenone) in pseudocereals (buckwheat, quinoa and amaranth) has been developed and validated by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS), using a QuEChERS-based sample treatment. This study also includes cereals such as spelt and white, brown and red rice. Matrix-matched calibration curves were established and limits of quantification were below the maximum contents established by EU regulation in cereals. The precision (repeatability and intermediate precision) was lower than 12% in all cases and recoveries were between 60.0% and 103.5%, fulfilling the current legislation. Finally, the content of aflatoxin B1 found in a red rice sample was confirmed by comparison of the result obtained by using immunoaffinity columns for extraction and clean-up.

Posted in Mass spectrometry, Pseudocereals, QuEChERS, Rice, Spelt, UHPLC | Tagged | Comments Off on Simple methodology for the determination of mycotoxins in pseudocereals, spelt and rice

Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence detection

Posted on 3 September, 2013 by fqm302

This paper presents two sample treatments, dispersive liquid–liquid microextraction (DLLME) and QuEChERS for the determination of 9 sulfonamides, regulated by the EU Council in milk samples. Both methods represent useful alternatives to conventional procedures based mainly in solid-phase extraction, in terms of simplicity, reduction of organic solvents, sample throughput and effectiveness for cleaning-up complex samples. They have been evaluated and compared in terms of efficiency, trueness, sensitivity and precision, using HPLC with fluorescence detection employing a previous derivatisation step with fluorescamine. Clean extracts were obtained with recoveries between 90.8–104.7% and 83.6–104.8% for DLLME and QuEChERS, respectively. Matrix-matched calibration curves were established for both methods using milk samples spiked at four concentration levels. LODs (3xS/N) lower than 1.21 µg/L and 2.73 µg/L for DLLME and QuEChERS, respectively, were obtained in all cases. The precision, in terms of repeatability and intermediate precision, was lower than 10% in all cases.

Posted in DLLME, Fluorescence, Milk, QuEChERS, Sulfonamides | Tagged | Comments Off on Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence detection

Ion-paired extraction of cephalosporins in acetone prior to their analysis by capillary liquid chromatography in environmental water and meat samples

Posted on 2 August, 2013 by fqm302

Ion-pair extraction of cephalosporins from aqueous solution into acetone by the addition of ammonium sulfate to a 1:2 (v/v) acetone–water solvent was carried out followed by their determination using reversed-phase capillary liquid chromatography. The analytes included are cephoperazone, cefquinome, cephalexin, cephapirin, cephaloniun, cephamandole, cephazolin and cephadroxile. In order to form the ion-pair, hexadecyltrimethylammonium bromide (CTAB) was selected as cationic ion-pairing agent at a concentration of 0.9 mM using 10 mM phosphate buffer at pH 8 as the optimum condition for the aqueous solution. The applied methodology, named salting-out assisted liquid/liquid extraction (SALLE) involves the use of 1.25 g of ammonium sulfate as salting-out agent.

The separation of cephalosporins using a Luna C18 (150 mm×0.3 mm, 5 µm, 100 Å) column was achieved under the following conditions: a gradient program combining solvent A (0.1% formic acid in water, pH 4) and solvent B (acetonitrile–methanol (50:50, v/v)), at a flow rate of 20 µl min−1, column temperature 35 °C and injection volume 7 µl with UV detection at 250 nm. The limits of quantification for the studied compounds were between 4.3 and 22.7 μg/L for water samples and 4.1 and 73.3 μg/kg in the case of beef samples, lower than the maximum residue limits permitted by the EU for this kind of food. The developed methodology has demonstrated its suitability for the analysis of these widely applied antibiotics in environmental water and meat samples, including beef and pork muscle, with high sensitivity, precision and satisfactory recoveries.

Posted in Capillary HPLC, Cephalosporins, Meat, SALLE, UV-vis, Water | Tagged , | Comments Off on Ion-paired extraction of cephalosporins in acetone prior to their analysis by capillary liquid chromatography in environmental water and meat samples

Micotoxinas: aproximaciones analíticas y metabolómicas

Posted on 26 July, 2013 by fqm302

Las micotoxinas son metabolitos secundarios tóxicos, de composición variada, producidos por diferentes hongos. Suelen contaminar alimentos, piensos, o las materias primas utilizadas para su elaboración, pudiendo afectar a la salud tanto de humanos como de animales. Debido a su gran variedad de efectos tóxicos, y sobre todo a su extrema resistencia al calor (termorresistencia), la presencia de micotoxinas en los alimentos es considerada de alto riesgo.

La contaminación de los alimentos con micotoxinas depende de las condiciones ambientales, que pueden propiciar el crecimiento del hongo y por ende la producción de las toxinas. Por tanto, 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.

Como resultado, la legislación a nivel europeo sobre seguridad alimentaria es cada vez más restrictiva en cuanto a los niveles de micotoxinas en alimentos y ha establecido contenidos máximos de estos compuestos que no deben ser superados con objeto de garantizar la calidad del producto y permitir su distribución y consumo.

Considerando las recientes e importantes mejoras de las técnicas separativas en cuanto a miniaturización y aumento de la eficacia, y con objeto de explorar sus indudables ventajas (bajo consumo de disolventes, alta sensibilidad, elevada resolución, tiempos de análisis reducidos y baja cantidad de muestra), en esta Tesis Doctoral se proponen nuevos métodos analíticos para la determinación de micotoxinas empleando técnicas de separación miniaturizadas (CE y HPLC capilar) y de alta eficacia (UHPLC) con objeto de explorar las ventajas mencionadas. Estas técnicas, acopladas a sistemas de detección altamente sensibles y selectivos, como LIF o MS/MS, permiten la cuantificación e identificación inequívoca de estos compuestos a las bajas concentraciones esperadas en estas matrices.

Por otro lado, es de gran importancia disponer de técnicas que permitan el estudio del metaboloma de los hongos implicados en la producción de micotoxinas y otros metabolitos secundarios, para conocer los genes productores o para determinar nuevos metabolitos, lo que podría ayudar a desarrollar estrategias para evitar la aparición de micotoxinas. En esta Tesis, se han empleado HRMS y MSn para el estudio del metaboloma de varios hongos.

A continuación se describe brevemente el trabajo desarrollado:

  1. En el primer capítulo se describe el método desarrollado para la determinación de las aflatoxinas B1, B2, G1 y G2 en muestras de arroz utilizando la cromatografía capilar electrocinética micelar (MEKC) con detección LIF y preconcentración
    online.

  2. En el segundo capítulo se ha llevado a cabo una comparación de diversos tratamientos de muestra para la determinación de ocratoxina A en diferentes tipos de vino, como son la microextracción líquido – líquido dispersiva (DLLME) empleando disolventes orgánicos y líquidos iónicos como extractantes (IL-DLLME), y la metodología QuEChERS (acrónimo formado a partir de los términos en inglés: Rápido, Eficaz, Barato, Fácil, Robusto, Seguro). En este caso, la técnica instrumental empleada ha sido la HPLC capilar – LIF, añadiendo a la fase móvil un medio micelar aniónico para aumentar la intensidad de la fluorescencia y mejorar la eficacia.

  3. En el capítulo tercero se ha llevado a cabo la determinación de 15 micotoxinas (incluyendo aflatoxinas, fumonisinas, tricotecenos, ocratoxina A, citrinina, esterigmatocistina y zearalenona) en cardo mariano (Silybum marianum). Para ello, se ha utilizado UHPLC – MS/MS para el análisis instrumental, mientras que el tratamiento de la muestra consistió en un primer paso basado en el procedimiento QuEChERS, que permitía la determinación de 5 micotoxinas (fumonisina B1, fumonisina B2, nivalenol, deoxinivalenol y fusarenona -X) y una posterior limpieza basada en la DLLME para la determinación del resto de las micotoxinas.

  4. Para demostrar el potencial de la UHPLC – MS/MS junto con el tratamiento de muestra QuEChERS, en el cuarto capítulo se han desarrollado y caracterizado dos métodos sensibles, simples y rápidos para la determinación de 15 micotoxinas (aflatoxina B1, aflatoxina B2, aflatoxina G1, aflatoxina G2, ocratoxina A, fumonisina B1, fumonisina B2, nivalenol, deoxinivalenol, fusarenona – X, toxina T-2 y HT-2, citrinina, esterigmatocistina y zearalenona) en cereales (espelta, arroz blanco, arroz integral y arroz rojo) y pseudocereales (trigo sarraceno, quinoa y amaranto) y de 10 micotoxinas (ocratoxina A, fumonisina B1, fumonisina B2, deoxynivalenol,
    fusarenona – X, toxina T-2 y HT-2, citrinina, esterigmatocistina y zearalenona) en melazas (de arroz, trigo y cebada).

  5. El capítulo quinto describe un método de análisis para la determinación de 14 micotoxinas en muestras de frutos secos (almendras, cacahuetes, semillas de girasol, semillas de calabaza, nueces, nueces de macadamia, pistachos, avellanas y piñones). De forma similar al capítulo tercero, el tratamiento de muestra comprende una primera etapa basada en el procedimiento QuEChERS para la determinación de las fumonisinas B1 y B2, deoxinivalenol, fusarenona – X, toxinas T-2 y HT-2, citrinina, esterigmatocistina, zearalenona y ocratoxina A y una posterior etapa de limpieza basada en la DLLME para la determinación de las aflatoxinas (B1, B2, G1 y G2).

  6. En capítulo sexto, se ha desarrollado un nuevo enfoque basado en cromatografía de líquidos (LC) acoplada a HRMS y LC-MSn, para la identificación de alcaloides ergóticos nuevos o poco explorados en muestrasde cereales.

  7. Finalmente, en el último capítulo se han determinado los metabolitos producidos por el cluster 27 pks (pks27) en el Aspergillus flavus usando LC-HRMS y LC-MSn. Asimismo, se ha estudiado por primera vez el patrón de fragmentación de estos compuestos usando ionización por electrospray en modo negativo. Este trabajo, junto con el correspondiente al capítulo sexto, han sido llevados a cabo en la Facultad de Ciencias Farmacéuticas de la Universidad de Gante (Bélgica).
Posted in Theses | Comments Off on Micotoxinas: aproximaciones analíticas y metabolómicas