Analysis of lamotrigine and lamotrigine 2-N-glucuronide in guinea pig blood and urine by reserved-phase ion-pairing liquid chromatography

Lamotrigine is an investigational anticonsulvant drug undergoing clinical trials. A simultaneous assay was developed to quantitate lamotrigine and its major metabolite, lamotrigine 2-N-glucuronide, from guinea pig whole blood. The extraction procedure and reversed-phase high-performance liquid chromatographic (HPLC) assay employed sodium dodecylsulfate (SDS) as an ion-pairing reagent to selectively separate lamotrigine and lamotrigine 2-N-glucuronide from endogenous blood components, other anti-convulsant drugs, and their metabolites. The mobile phase was composed of acetonitrile-50 mM phosphoric acid (pH 2.2) containing 10 mM SDS (33:67, v/v), and components were detected at 277 nm. The total coefficients of variance (C.V.) for the blood assay were ≤ 9.4% for lamotrigine (0.25-20.0 μg/ml) and ≤ 13.4% for the glucuronide metabolite (0.25-10.0 μg/ml). Separate assays for lamotrigine and its glucuronide in urine were developed. In order to quantitate low levels of lamotrigine in guinea pig urine, lamotrigine was extracted with tert.-butyl methyl ether-ethyl acetate (1:1). The total C.V. for lamotrigine quantitation in urine was ≤ 7.5% (0.10-10.0 μg/ml). For the determination of lamotrigine 2-N-glucuronide, urine was diluted with an SDS-phosphoric acid buffer (1:4) and injected directly onto the HPLC system, total C.V. ≤ 4.2% (0.5-50 μg/ml).