Determinación cuantitativa de fenobarbital en plasma sanguíneo mediante espectrometría UV
Fecha
2017-07
Autores
Sánchez García, Daniel
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Editor
Universidad Central “Marta Abreu” de Las Villas. Facultad de Química Farmacia. Departamento de Licenciatura en Química
Resumen
En este trabajo se propone un método de cuantificación de fenobarbital en muestras de plasma sanguíneo para aplicación en toxicología forense. Este método procede a través de tres etapas fundamentales: precipitación ácida de las proteínas; ELL y determinación mediante espectrofotometría UV. El método para la determinación espectrofotométrica se fundamenta en el hecho de que la absorción UV de las disoluciones acuosas de barbitúricos está íntimamente ligada a su pH y sus formas no ionizadas solo absorben muy débilmente. Se estudiaron las condiciones para la precipitación de las proteínas comparando diferentes agentes precipitantes, el mejor resultado se obtuvo con HCl al 35 %. Se realizaron dos ELL con éter dietílico de forma sucesiva, se realizó un lavado del extracto etéreo con buffer de tetraborato de sodio (Na2B4O7) y colectó el extracto final en hidróxido de sodio (0,45 mol/L). Se determinó el FR de la extracción para diferentes concentraciones del analito y se comprobó la correlación entre el recobrado y la concentración de fenobarbital en plasma a concentraciones entre 10 y 50 mg/L, no así para concentraciones menores que 10 mg/L. El estudio mostró que la concentración del analito debe expresarse en función de las diferencias de absorbancias medidas a 254 nm y en condiciones de pH 13 y 2 respectivamente. Se determinó, bajo estas condiciones, que la curva patrón cumple con los parámetros de linealidad y correlación. La concentración determinada en muestras de concentración conocida siguiendo el método propuesto mostró un error de solo el 0,99 %.
In this paper, we propose a method of quantification of phenobarbital in blood plasma samples for application in forensic toxicology. This method proceeds through three fundamental stages: acid precipitation of proteins; LLE and determination by UV spectrophotometry. The method for the spectrophotometric determination is because the UV absorption of the aqueous solutions of barbiturates is intimately linked to its pH and its non-ionized forms only absorb very weakly. The conditions for precipitation of proteins were studied by comparing different precipitating agents; the best result was obtained with HCl (35 %). Two LLE were carried out with diethyl ether successively, a washing of the ethereal extract was performed with sodium tetraborate buffer (Na2B4O7) and the final extract was collected in sodium hydroxide (0.45 mol/L). The extraction RF was determined for different concentrations of the analyte and the correlation between the recovery and the concentration of phenobarbital in plasma at concentrations between 10 and 50 mg/L was verified, but not for concentrations below 10 mg/L. The study showed that the concentration of the analyte should be expressed as a function of the absorbance differences measured at 254 nm and under conditions of pH 13 and 2 respectively. It was determined, under these conditions; the standard curve complies with the parameters of linearity and correlation. The concentration determined in known concentration samples following the proposed method showed an error of only 0.99 %.
In this paper, we propose a method of quantification of phenobarbital in blood plasma samples for application in forensic toxicology. This method proceeds through three fundamental stages: acid precipitation of proteins; LLE and determination by UV spectrophotometry. The method for the spectrophotometric determination is because the UV absorption of the aqueous solutions of barbiturates is intimately linked to its pH and its non-ionized forms only absorb very weakly. The conditions for precipitation of proteins were studied by comparing different precipitating agents; the best result was obtained with HCl (35 %). Two LLE were carried out with diethyl ether successively, a washing of the ethereal extract was performed with sodium tetraborate buffer (Na2B4O7) and the final extract was collected in sodium hydroxide (0.45 mol/L). The extraction RF was determined for different concentrations of the analyte and the correlation between the recovery and the concentration of phenobarbital in plasma at concentrations between 10 and 50 mg/L was verified, but not for concentrations below 10 mg/L. The study showed that the concentration of the analyte should be expressed as a function of the absorbance differences measured at 254 nm and under conditions of pH 13 and 2 respectively. It was determined, under these conditions; the standard curve complies with the parameters of linearity and correlation. The concentration determined in known concentration samples following the proposed method showed an error of only 0.99 %.
Descripción
Palabras clave
Método de Cuantificación, Plasma Sanguíneo, Fenobarbital, Espectrofotometría UV