Simultaneous determination of six analytes by HPLC-UV for high throughput analysis in permeability assessment

A methodology for the simultaneous determination of six control analytes, including carbamazepine, desipramine, guanabenz, methotrexate, propranolol, and warfarin, was developed and validated utilizing reversed-phase high-performance liquid chromatography with ultraviolet detection for high throughput analysis for permeability assessment. The analytes were separated on Agilent Zorbax SB-C18 (50 × 4.6 mm I.D., 5 μm) with a gradient mobile phase consisting of water (containing 1% isopropyl alcohol and 0.01% heptafluorobutyric acid) and acetonitrile (containing 1% isopropyl alcohol and 0.01% heptafluorobutyric acid). The flow rate was 2.0 mL/min and the eluent was monitored at 280 nm. A linear response was found for all six analytes over a broad concentration range (1.00-200 μM). The correlation coefficient for each analyte was greater than 0.999. The limit of detection and limit of quantitation were 0.03 and 0.10 μM, 0.10 and 0.30 μM, 0.05 and 0.15 μM, 0.03 and 0.10 μM, 0.05 and 0.15 μM, 0.10 and 0.30 μM for carbamazepine, desipramine, guanabenz, methotrexate, propranolol, and warfarin, respectively. The optimized method was further successfully applied to high throughput analysis for parallel artificial permeability assay.