Validación de métodos analíticos por cromatografía líquida alta resolución (HPLC) para la cuantificación de cefuroxima, delafloxacino y tilvalosina en muestras biológicas

Resumen

The use of antibiotics in both human and veterinary medicine is currently being restricted due to the high resistance offered by microorganisms. Pharmacokinetic studies are a very valuable tool to assess drug concentration in the body and to establish appropriate dosages in order to ensure clinical success and minimize the risk of the appearance of bacterial resistance. In order to carry out these studies on biological samples, both from humans and animals, it is necessary first to develop and validate an analytical method to adequately quantify drug concentrations in the sample. In general, the analytical methods most used in pharmacokinetic studies for drug quantification are chromatographic methods. In this case, the general objective of this doctoral thesis was the validation of three analytical methods for the quantification of three antibiotics (cefuroxime, delafloxacin, tylvalosin) that allows to obtain exact concentrations of the drug in different biological matrices and thus use them to perform relevant pharmacokinetic studies. The development, optimization and validation of an analytical method for the quantification of cefuroxime was carried out in human plasma by HPLC/UV. Extraction was performed by protein precipitation with methanol/TFA (trifluoroacetic acid). Validation parameters were satisfactory, obtaining a high recovery (93.52%), high selectivity and specificity, with an accuracy error value below 10%. Similarly, for the calculation of precision, a value ˂ 10% was obtained in the coefficient of variation. Detection and quantification limits were 0.1 and 0.25 µg/mL, respectively. Therefore, the proposed analytical method is suitable to be applied in clinical studies, routine analysis and pharmacokinetic studies. The development, optimization and validation of an analytical method for the quantification of delafloxacin was performed in human plasma by HPLC/FL. The extraction process consisted of a liquid-liquid extraction with 50% formic acid and ethyl acetate. The stability of the antibiotic in different matrices and at different temperatures was also evaluated. Validation parameters were satisfactory, obtaining a high recovery (98.3%), high selectivity and specificity, a detection limit of 0.05 µg/mL and a quantification limit of 0.1 µg/mL. The values obtained in precision, expressed as coefficient of variation, were ˂ 11%. Similarly, the values obtained for the accuracy error were ˂ 11%. Stability studies showed instability in both plasma samples and mother solutions after fifteen days of storage at -40ºC. Hence, new studies are needed to ensure the stability of delafloxacin in different storage conditions. The development, optimization and validation of an analytical method for the quantification of tylvalosin was carried out in pig plasma by HPLC/UV. Extraction was performed by protein precipitation with a 0.1% solution of formic acid in acetonitrile. Antibiotic stability in different matrices and at different temperatures was also evaluated. Validation parameters were excellent, obtaining a range in the recovery value between 89.66-96.92%. The detection and quantification limits were 0.05 μg/mL and 0.1 μg/mL, respectively. Regarding the values obtained for precision and accuracy error, these were both lower than 13.0%. The good selectivity and specificity of this analytical method should be highlighted. Finally, the results obtained in the stability studies at -40ºC were also satisfactory, demonstrating short- and long-term stability. Therefore, it can be concluded that this method can be applied in clinical studies, effective levels monitoring and pharmacokinetic studies.