A Liquid-Liquid Extraction-Based Validation Method for the Quantification of Levetriracetam in Human Plasma

Abstract

Levetiracetam in human plasma was quantified using a simple, sensitive, and fast high performance liquid
chromatography approach with UV detection (215 nm). An isocratic mobile phase consisting of a mixture of
buffer (5mM Di-Potassium hydrogen phosphate anhydrous, pH7.2): Methanol (85:15 v/v) on a reverse phase
C-8 Kromasil column was used to separate the analyte and internal standard (Zonisamide) after a single-step
liquid-liquid extraction with diethyl ether/dichloromethane (70/30 v/v). An absolute standard deviation below
20% and a quantization cutoff of 1μg/mL were required. An established linear range was found to be between
1μg/mL and 40μg/mL. The validated HPLC technique achieved between-batch accuracy of 5.6-8.9% and
within-batch precision of 3.9-5.3%. The accuracy ranged from 99.9 to 106.3% across batches, and from 96.1
to 102.0% within them. Medications given at the same time usually had no effect on the procedures outlined.
Levetiracetam exhibited >90% plasma stability, with no signs of deterioration throughout autosampler sample
processing or 60 days of freezer storage. This approach has been tested and shown to be both sensitive and easy
to use in pharmacokinetic research, and it has a between-batch accuracy of less than 10%.