Determination of Pregabalin by HPLC with Precolumn Derivatization Using 1-Fluoro-2,4-dinitrobenzene

A high performance liquid chromatographic (HPLC) method with UV detection was developed to determine the content of Pregabalin (PGB) by using 1-fluoro-2,4-dinitrobenzene (DNFB) as the precolumn derivatization reagent. The effects of light, reaction temperature, molar ratio of DNFB to PGB and reaction time on the derivatization reaction were investigated. Chromatographic separation was achieved on a C18 column with a mixture of phosphate buffer and acetonitrile as the mobile phase. The relationship between the PGB concentration and the PGB-DNFB peak area on the chromatograph is linear over the PGB concentration range of 8～48 μg·mL-1 (r = 0.9998). The recover ratio is (100.03 ± 1.10)% (n = 9). The within-day precisions are 0.82%, 0.77% and 1.87% for three different PGB concentrations (n = 6), respectively. The derivative solution is stable within 8 hours (RSD = 1.92%). The LOD is as low as 0.8 ng·mL-1. The proposed PGB content determination method is simple, rapid, accurate and reliable.