Determination of levetiracetam enantiomeric purity using HPLC-fluorescence detection coupled with a pre-column ophthalaldehyde-N-acetyl-L-cysteine derivatization

A simple and cost effective method was developed for enantiomeric separation of levetiracetam (LEV) and chiral impurity R-enantiomer in raw material and tablets. The method based on derivatization of racemate using o-phthalaldehyde and N-acetyl-L-cysteine in basic borate buffer. The diastereomeric isoindole derivatives were separated by HPLC efficiently using C18 column and ammonium acetate (20 mM, pH: 7.3): methanol (55:45) as mobile phase, and isocratic elution at a flow rate of 1.0 mL/min. The optimal fluorescence detection parameters were λex = 330 nm, and λem = 450 nm. The method was validated and applied to raw material and tablet samples. Enatiomeric resolution was achieved within 9 minutes, and the resolution factor was more than 2. Calibration curves were linear over the range 1-50 and 0.06-8.0 (µg/mL) for LEV and R-enantiomer, respectively. LOD and LOQ of R- enantiomer were 0.02, and 0.06 (µg/mL), respectively. The method was sensitive, accurate, and precise. The developed method had been applied successfully to determine R-enantiomer in LEV raw material and tablets and can be used in quality control laboratories.

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