Authors
Abstract(s)
A taxa de incidência de epilepsia na maioria dos países desenvolvidos é, hoje
em dia, de cerca de 40-70 por 100 000 indivíduos. As crises epilépticas são
normalmente controladas com auxílio dos fármacos antiepilépticos, estando
disponível um grande número de agentes terapêuticos. A possibilidade real de
ocorrerem interacções entre estes fármacos, o advento da internet e o problema
crescente da automedicação podem levar a um aumento considerável do
número de intoxicações devidas a estes compostos.
Deste modo, são necessários métodos analíticos rápidos e sensíveis para a
quantificação fidedigna destes compostos em amostras biológicas humanas,
para dar auxílio e assistência aos serviços de emergência no tratamento de
indivíduos intoxicados.
O objectivo deste trabalho foi então o desenvolvimento e validação de um
método para a determinação quantitativa de quatro fármacos antiepilépticos
(primidona, carbamazepina, fenitoína e fenobarbital) e o metabolito da
carbamazepina (10,11-epoxi-carbamazepina) em plasma humano, utilizando
extracção em fase sólida e cromatografia líquida de alta eficiência acoplada a um
detector de fotodiodos (HPLC-DAD).
Utilizando um volume de amostra de apenas 0,2 mL, o método foi
completamente validado de acordo com normas internacionalmente aceites para
a validação de métodos bioanalíticos. Os parâmetros estudados foram
selectividade, linearidade, limites de detecção (LOD) e de quantificação (LOQ),
precisão e exactidão, estabilidade em amostras processadas e em ciclos de
congelação/descongelação, e eficiência de extracção. O método foi selectivo, já que não foram detectados interferentes após análise de amostras brancas de plasma de 20 origens diferentes, tendo sido obtida uma resposta linear entre 0.25 e 25 μg/mL para todos os compostos, excepto para a carbamazepina (0.1-25 μg/mL), primidona (1-25 μg/mL) e fenitoína (0,5-25 μg/mL). Os valores obtidos para a precisão e exactidão intra- e interdia estavam em conformidade com os critérios de validação mencionados, i.e., coeficientes de variação inferiores a 15% e bias dentro de um intervalo de ±15% da concentração teórica (excepto no limite inferior de quantificação, para o qual 20% foi considerado adequado). A eficiência de extracção foi superior a 90% para todos os compostos.
Finalmente, o método foi aplicado a quatro casos de intoxicação, nos quais alguns destes fármacos estavam presentes.
Em conclusão, o método desenvolvido provou ser útil para a determinação destes fármacos antiepilépticos em situações de intoxicação clínica ou forense.
Nowadays, in most developed countries, epilepsy’s incidence rate is of about 40- 70 in 100000. Epileptic seizures are controlled by means of antiepileptic drugs, and a number of therapeutic agents are available. The possibility of drug interactions between these agents, the advent of internet and the growing problem of self-medication may lead to an increased number of intoxications. Therefore, there is the need of rapid and sensitive analytical methods for the reliable quantitation of these compounds in human biological samples to help and assist emergency services in the treatment of intoxicated individuals. The goal of this work was the development and validation of a method for the quantitative determination of four selected antiepileptic drugs (primidone, carbamazepine, phenytoin and phenobarbital) and metabolite (10,11- epoxycarbamazepine) in human plasma, using solid-phase extraction and liquidchromatography-diode array detector (HPLC-DAD). The method used a sample amount as low as 0.2 mL, and was fully validated according to internationally accepted guidelines for bioanalytical method validation. The studied parameters were selectivity, linearity, limits of detection (LOD) and quantitation (LLOQ), precision and accuracy, bench top and freeze/thaw stability, and extraction efficiency. The method was selective, as no interfering compounds were detected by analysis of blank plasma samples of 20 different origins, and linear from 0.5-25 µg/mL for all compounds, except for carbamazepine (0.25-25 µg/mL) and primidone (1-25 µg/mL). Intra- and interday precision and accuracy were in conformity with the above mentioned criteria, i.e., coefficients of variation of less than 15% and bias within a ±15% interval from the nominal value (excepted at the LLOQ, for which 20% was accepted). Extraction efficiency was higher than 90% for all compounds. Finally, the method was successfully applied to four cases of intoxication with some of these compounds. In conclusion, the developed method was proven useful for the determination of these antiepileptic drugs, both in clinical and forensic toxicology scenarios.
Nowadays, in most developed countries, epilepsy’s incidence rate is of about 40- 70 in 100000. Epileptic seizures are controlled by means of antiepileptic drugs, and a number of therapeutic agents are available. The possibility of drug interactions between these agents, the advent of internet and the growing problem of self-medication may lead to an increased number of intoxications. Therefore, there is the need of rapid and sensitive analytical methods for the reliable quantitation of these compounds in human biological samples to help and assist emergency services in the treatment of intoxicated individuals. The goal of this work was the development and validation of a method for the quantitative determination of four selected antiepileptic drugs (primidone, carbamazepine, phenytoin and phenobarbital) and metabolite (10,11- epoxycarbamazepine) in human plasma, using solid-phase extraction and liquidchromatography-diode array detector (HPLC-DAD). The method used a sample amount as low as 0.2 mL, and was fully validated according to internationally accepted guidelines for bioanalytical method validation. The studied parameters were selectivity, linearity, limits of detection (LOD) and quantitation (LLOQ), precision and accuracy, bench top and freeze/thaw stability, and extraction efficiency. The method was selective, as no interfering compounds were detected by analysis of blank plasma samples of 20 different origins, and linear from 0.5-25 µg/mL for all compounds, except for carbamazepine (0.25-25 µg/mL) and primidone (1-25 µg/mL). Intra- and interday precision and accuracy were in conformity with the above mentioned criteria, i.e., coefficients of variation of less than 15% and bias within a ±15% interval from the nominal value (excepted at the LLOQ, for which 20% was accepted). Extraction efficiency was higher than 90% for all compounds. Finally, the method was successfully applied to four cases of intoxication with some of these compounds. In conclusion, the developed method was proven useful for the determination of these antiepileptic drugs, both in clinical and forensic toxicology scenarios.
Description
Keywords
Epilepsia Carbamazepina Fenitoína Fenobarbital Antiepilépticos - Cromatografia líquida Fotodiodos