Development and validation of a quantitative method for determination of retigabine and its N-acetyl metabolite; overcoming challenges associated with circulating labile N-glucuronide metabolites

Abstract

Retigabine or ezogabine is an anticonvulsant approved for use in adjunctive treatment of partial epilepsy in adults. In humans, there is no evidence for cytochrome P450 catalyzed reactions; the drug is extensively metabolized primarily by N-glucuronidation reactions and acetylation to form a mono-N-acetylated metabolite. The majority of the drug-related material has been found to be excreted in the urine. The major challenge in developing a method for the quantitation of retigabine in human plasma or urine is minimizing the contribution from labile N-glucuronides, which are known to circulate at very high levels (approximately 25-fold) relative to parent. Degradation of these metabolites during sample handling and processing has been shown to lead to an increase in the concentrations of both retigabine and the N-acetyl metabolite; where this conversion is temperature, pH, and time dependent. Thus, it is important to consider these observations while developing a method for the accurate quantitation of retigabine from biological matrices in order to prevent the overestimation of both retigabine and its N-acetyl metabolite. Herein, we describe an extraction procedure to ensure accurate quantitation of retigabine and its N-acetyl metabolite from human plasma. This publication also provides specific recommendations for sample handling and storage of clinical samples prior to bioanalysis. The method was validated in human plasma over the concentration range of 5–2500 ng mL⁻¹ for both analytes; the results from assay validation and incurred sample reproducibility demonstrate the method is rugged, precise, accurate, and well-suited for quantitative bioanalysis of both the drug and main metabolite.