Totally Automated Analysis by Robotized PrepStation and Liquid Chromatography: Direct-Sample Analysis of Felbamate

Abstract

A totally automated analysis of felbamate was developed by using a robotized PrepStation for extraction, followed by automated liquid chromatographic (LC) analysis and data reduction. This is one of the newer direct-sample analysis approaches by LC. Felbamate was a previously approved antiepileptic agent used to treat partial seizures with and without generalization and to treat Lennox-Gastaut syndrome in pediatric patients. However, due to the reported incidences of aplastic anemia, its clinical application was recently restricted to the treatment of the latter syndrome. The automated assay using Bench Supervisor, PrepStation, and LC, based on a previously developed manual method, used 200 microliters of serum standards, quality control, or patients' plasma. These were mixed with 600 microliters of internal standard (IS) W509 dissolved in acetonitrile for protein precipitation. After axial centrifugation and standing, aliquots of the clear supernatant were transferred and washed with hexane. Aliquots of the supernatant were transferred and injected into a high-performance liquid chromatograph (HPLC). HPLC parameters included an mu Bondapak C-18 column, phosphate/acetonitrile (8:2) as mobile phase, and detection at 214 nm. Retention times were 2.9 and 4.2 min for felbamate and IS, respectively. Calibration was linear for concentrations from 10 to 200 mg/L with r > 0.994. Precision studies showed coefficients of variation ranging from 2.7% to 8.8%. Correlation with the manual method showed that r = 0.934, slope = 1.048, intercept = -2.642, and n = 21. Phenobarbital coeluted with the IS. This study demonstrated the feasibility of using a robotized, automated method for monitoring felbamate, readily extended to monitoring other antiepileptic drugs with minimal modification.