Liquid chromatography–mass spectrometric determination of rufinamide in low volume plasma samples

Quantification of rufinamide in plasma was achieved using a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method. The chromatographic separation was achieved on a reversed phase column (Zorbax SB-C18 100 mm × 3 mm, 3.5 μm) under isocratic conditions. The mobile phase consisted of a mixture of water containing 0.1% formic acid and methanol (50:50, v/v). The mass spectrometric detection of the analyte was in multiple reaction monitoring mode (MRM) using an electrospray positive ionization (ESI positive). The monitored ions were 127 m/z derived from 239 m/z rufinamide and 108 m/z derived from 251 m/z the internal standard (lacosamide). Protein precipitation with methanol was applied for sample preparation using only 50 μl aliquots. The concentration range was 40–2000 ng/ml for rufinamide in plasma. The limit of detection was 1.25 ng/ml and the lower limit of quantification was established at 5 ng/ml rufinamide concentration. Selectivity and matrix effect was verified using individual human, rat and rabbit plasma samples. Short-term, post-preparative and freeze–thaw stability was also investigated. The proposed method provides accuracy, precision and high-throughput (short runtime 4.5 min) for quantitative determination of rufinamide in plasma. This is the first reported liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for analysis of rufinamide from low volume plasma samples. The LC–MS/MS method was validated according to the current official guidelines and can be applied to accurately measure rufinamide level of large number of plasma samples from clinical studies or therapeutic drug monitoring.