Development and validation of a HPLC-UV assay for quantification of levetiracetam concentrations in critically ill patients undergoing continuous renal replacement therapy.

ABSTRACT

Limited clinical data exists on the effects of continuous renal replacement therapy (CRRT) on drug pharmacokinetics. A high-performance liquid chromatography with ultraviolet detection method was developed and validated to determine levetiracetam concentrations in human plasma and CRRT effluent samples. Five hundred microliters of human plasma and 250 µL effluent samples were used to quantify levetiracetam. Plasma samples were purified by protein precipitation, evaporated under nitrogen gas at room temperature and reconstituted in 50 mm potassium dihydrogen phosphate buffer (pH of 4.5). Reverse-phase chromatographic separation was achieved within 20 min using a mobile phase eluting gradient of 50 mm potassium dihydrogen phosphate and acetonitrile. UV detection was set at 195 nm. The calibration curve was found to be linear over the range of 2-80µg/mL. Inter- and intra-day precisions were < 8% for both plasma and effluent samples. The accuracy was determined to be within -12-10% of nominal concentrations. The method was selective and sensitive with a lower limit of quantification of 2 µg/mL. Overall recovery of levetiracetam from plasma was ~100%. The validated assay was successfully applied in a pharmacokinetic study to determine potential dose adjustments in patients undergoing CRRT and receiving levetiracetam.