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PUBLICACION 1

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Este es un artículo del que puedes obtener la versión en .pdf de forma gratuita:

http://journals.tubitak.gov.tr/chem/issues/kim-02-26-4/kim-26-4-8-0106-13.pdf

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PUBLICACION 2

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Distribución de los niveles de cafeína en orina según diferentes deportes antes y después de quitarla de la lista prohibida de la Agencia Mundial Antidopaje

Resumen en español: La cafeína es la droga más comúnmente utilizada hoy en día en todo el mundo. Diferentes estudios han descrito la influencia positiva de la cafeína en el rendimiento (Graham et al., 1994; Sinclair & Geiger, 2000; Tarnopolsky, 1994) de deportes de larga duración y también en los de corta duración, aunque en este caso los efectos no son tan claros. Lo que sí está admitido es que los efectos sobre la marca se observan cuando las dosis utilizadas son de entre 3 y 6 miligramos de cafeína por kilogramo de peso corporal, porque cuando se utilizan dosis mayores, los efectos sobre la marca deportiva no son mejores e, incluso, pueden ser perjudiciales. Como estas dosis de 3 a 6 miligramos por kilogramo de peso, son las dosis de cafeína que habitualmente suelen tomar los consumidores habituales de café, la WADA decidió suprimir la cafeína de las lista prohibida desde el 1 de enero de 2004. Sin embargo, los laboratorios de control de dopaje siguen analizando la cafeína en las muestras de orina, para ver si el hecho de haberla eliminado de la lista prohibida provoca un aumento desmesurado de su utilización. En un estudio realizado por W. Van Thuyne y col., que trabajan en el Laboratorio de Control de Dopaje acreditado por la WADA del Departamento de Química Clínica, Micrología y Inmunología de la Universidad de Ghent (Bélgica), publicado en el número 27 de la revista científica International Journal of Sports Medicine del año 2006, los autores midieron la concentración de cafeína en orina de tests de dopaje de competición realizados después del 1 de enero de 2004 (cuando se suprimió la cafeína de la lista prohibida) a 4633 deportistas de Bélgica pero también de Holanda y de la Federación Internacional de Atletismo, de la Unión Ciclista Internacional y de la Unión Europea de Fútbol (UEFA). Los resultados de estos análisis se compararon con los de los resultados de los análisis que se habían realizado en ese mismo laboratorio entre 1993 y 2002, cuando la cafeína estaba prohibida y se consideraba y sancionaba como dopaje si su concentración en orina superaba 12 microgramos de cafeína por mililitro de orina. Los resultados de los análisis muestran que: 1) si la cafeína estuviese ahora prohibida, como ocurría antes de 2004, habría habido 6 resultados positivos declarados como dopaje (el 0.13% del total), porque sus concentraciones en orina superaron 12 microgramos por mililitro. Los resultados positivos se habrían dado en ciclismo (4), atletismo (1) y fisioculturismo (1), 2) las concentraciones medias de cafeína en orina, en toda la población analizada, no han aumentado desde que se ha suprimido la cafeína de la lista prohibida, 3) tanto antes de 2004, como después de 2004, los deportes que presentaban concentraciones más altas de cafeína en orina fueron el power lifting, el fisioculturismo y el ciclismo y 4) en la mayoría de los deportes, la concentración de cafeína en orina ha disminuido después de 2004, excepto en ciclismo y en gimnasia, en los que se ha observado un aumento.

Aplicaciones prácticas: Estos resultados indican que, en general, la supresión de la cafeína de la lista prohibida no se ha acompañado de un mayor abuso de su utilización. Sin embargo, parece conveniente seguir analizándola, especialmente en algunos deportes como el ciclismo, el power lifting, el fisioculturismo y la gimnasia en los que, o bien se utilizan clásicamente con frecuencia, o bien, como en el caso de la gimnasia, parece que se empiezan a utilizar más.

Estos resultados indican que, en general, la supresión de la cafeína de la lista prohibida no se ha acompañado de un mayor abuso de su utilización. Sin embargo, parece conveniente seguir analizándola, especialmente en algunos deportes como el ciclismo, el power lifting, el fisioculturismo y la gimnasia en los que, o bien se utilizan clásicamente con frecuencia, o bien, como en el caso de la gimnasia, parece que se empiezan a utilizar más.

Caffeine concentrations were measured in the urine of 4633 athletes tested for doping control in the Ghent Doping Control Laboratory in 2004. Determination of these concentrations was done using an alkaline extraction with a mixture of dichloromethane and methanol (9 : 1; v/v) followed by high performance liquid chromatography and ultraviolet detection (HPLC-UV). The method was validated according to ISO 17 025 standards (International Organisation for Standardisation). Quantification was done by using a linear calibration curve in the range from 0 to 20 µg/ml. The limit of quantification (LOQ) was 0.10 µg/ml. Because the results were not normally distributed, transformation of the data was done to evaluate the difference in detected concentrations in several sports. This resulted in an overall average concentration of 1.12 ± 2.68 µg/ml. Comparison of the most frequently tested sports in 2004 demonstrated that caffeine concentrations in samples originating from power lifters are significantly higher in comparison to urines taken in other sports. Also, a significant difference between caffeine concentrations found in cycling and concentrations found in other sports, including athletics and some ball sports, was observed. A comparison was made between results obtained in 2004 and results obtained before the removal of caffeine from the WADA (World Anti-Doping Agency) doping list indicating that average caffeine concentrations decreased after the withdrawal of caffeine from the list of prohibited substances. The overall percentage of positive samples between the two periods remained the same although the percentage of positive samples noticed in cycling increased after the removal of caffeine from the doping list.

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PUBLICACION 3

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A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine

Abu-Qare AW, Abou-Donia MB.

J Pharm Biomed Anal. 2001; 26(5-6):939-47

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

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PUBLICACION 4

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A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers

GHOSHEH O. A. (1) ; BROWNE D. (2) ; ROGERS T. (1) ; DE LEON J. (2) ; DWOSKIN L. P. (1) ; CROOKS P. A. (1) ;

Résumé / Abstract. A simple isocratic HPLC procedure has been developed for the quantification of caffeine and the nicotine metabolites cotinine, 3'-hydroxycotinine, cotinine glucuronide and 3'-hydroxycotinine glucuronide in the plasma of smokers. The glucuronide conjugates were determined indirectly via initial basic hydrolysis of the analyte sample followed by quantification of the resulting deconjugation product. Plasma was basified, extracted with dichloromethane, evaporated, the residue dissolved water and an aliquot part was analyzed by HPLC. The method utilized a Partisil-10 SCX cation-exchange column and an isocratic mobile phase of sodium phosphate buffer: methanol (92:8 V/V, 0.1 M, adjusted to pH 4.8 with triethylamine) at a flow rate of 1.5 ml/min. UV detection was at 254 nm. All solutes were separated with good resolution, and quantification was determined using an internal standard of N,N'-diethylnicotinamide The retention times were: caffeine 5.1 min, 3'-hydroxycotinine 7.2 min, N,N-diethylnicotinamide 9.5 min, and cotinine 15.5 min. Detection limits for caffeine, 3'-hydroxycotinine, cotinine, and total cotinine were 10 ng/ml; the detection limit for total 3'-hydroxycotinine was 20 ng/ml. The inter-day and intra-day variations for all analytes were between 1 and 8%. This analytical method is suitable for the determination of caffeine and nicotine metabolite levels in large numbers of clinical samples.Journal of pharmaceutical and biomedical analysis

Mots-clés anglais / English Keywords: Cotinine ; Caffeine ; Quantitative analysis ; HPLC chromatography ; Blood plasma ; Nicotine ; Drug of abuse ; Tobacco ; Metabolite ; Human ; Glucuroconjugate ;

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PUBLICACION 5

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Validation of a high-performance liquid chromatography assay for quantification of caffeine and paraxanthine in human serum in the context of CYP1A2 phenotyping

J. P. Koch 1 2, G. W. ten Tusscher 3, J. G. Koppe 3, H. J. Guchelaar 2 *

Biomedical Chromatography  1999, vol.13, pp. 309-314

Abstract. In this study the validation of a reversed-phase high-performance liquid chromatography (HPLC) method, with UV-detection, for both caffeine and paraxanthine in human serum is described. This method is feasible for cytochrome P450 1A2 (CYP1A2) phenotyping, according to the results of a pilot study. With this HPLC method caffeine and paraxanthine can be determined selectively and specifically. In the expected concentration range, caffeine recoveries were 98-108% (within-run variation 4.0-6.4%, between-run variation 6.4-8.8%), paraxanthine recoveries were 96.6-97.5% (within-run variation 5.0-7.2%, between-run variation 7.2-10.8%). The limits of detection for caffeine and paraxanthine using this HPLC system were 0.3 and 0.1 mg/L, respectively. Linear calibration curves for both caffeine and paraxanthine were obtained in the concentration range 0.5-30 mg/L (r > 0.9999. Serum samples were stable for a week, when stored at -20 and +4°C.

Stability studies of selected doping agents in urine: Caffeine

VENTURA R. (1 2) ; JIMENEZ C. (1 2) ; GLOSAS N. (1) ; SEGURA J. (1 2) ; DE LA TORRE R. (1 2)

(1) Unitat de Recerca en Farmacologia, Institut Municipal d'Investigació Mèdica (IMIM), Doctor Aiguader 80, 08003 Barcelona, ESPAGNE; (2) Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, ESPAGNE

Résumé / Abstract

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C 18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4 ± 6.0 and 87.3 ± 5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 μg/ml (r[2] > 0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 μg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 μg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 C and in liquid urine samples stored at 4, -20, -40 and -80°C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 C for 7 days. The effect of repeated freezing (at -20 C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.

Journal of chromatography. B (J. chromatogr., B) ISSN 1570-0232