Validation of a Low‐Cost Reversed‐phase High‐Performance Liquid Chromatography‐Ultraviolet Detection Method for Measuring Levetiracetam Levels in Patients With Epilepsy

ABSTRACT A high‐performance liquid chromatography (HPLC) method with detection at 195 nm was developed and fully validated for the quantification of levetiracetam in human plasma, following Food and Drug Administration bio‐analytical method validation guidelines. Plasma samples were obtained from blood collected in ethylenediaminetetraacetic acid tubes and prepared by protein precipitation with methanol prior to analysis. The method employed a reversed‐phase core‐shell column (2.6 µm, 100 Å, 75 × 4.6 mm), with ultraviolet (UV) detection at 195 nm and a flow rate of 1 mL/min from a 7 µL injection volume. The mobile phase, delivered via a quaternary pump, consisted of 90.0% pH‐adjusted 20.0 mM potassium dihydrogen phosphate buffer and 10.0% organic solvent, composed of methanol and acetonitrile in a 3:1 ratio. Calibration was linear over the 2.5–100 µg/mL range ( r = 0.9999). The relative standard deviation was below 5% for both intra‐ and inter‐day precision and accuracy, except at the lower limit of quantification (2.5 µg/mL), where inter‐day variability was 6.92%. Specificity was confirmed using patient samples, with no observed interference from 36 co‐administered drugs, three infusion solutions, or one dietary supplement. This study presents a fully validated, cost‐effective HPLC‐UV detection method developed using a newly applied short core‐shell column and an innovative three‐component mobile phase composition, enabling rapid and selective quantification of levetiracetam in plasma, even in polypharmacy settings.