Development and validation of an Enzyme Linked Immunosorbent Assay (ELISA) for gentamicin quantification in dried blood spot samples

Abstract

A competitive enzyme linked immunosorbent assay (ELISA) procedure has been developed for the quantification of gentamicin in dried blood spot (DBS) samples collected from paediatric patients on Guthrie cards. Gentamicin was extracted from DBS samples by vortexing with the ELISA extraction buffer for 30 minutes. The ELISA assay was successfully validated using ICH guidelines for assay validation over the concentration range of 0.15-2.5 μg/ml. The standard deviation, mean, coefficient of variation (CV%) and relative error (RE%) were evaluated and demonstrated good assay reproducibility. The validated ELISA method can be applied to clinical DBS samples. This assay will be utilised to quantify gentamicin in DBS samples collected from paediatric (premature neonatal) patients, in which the quantification data will be utilised for population pharmacokinetic studies of gentamicin in premature neonates.