Impurity profile study of lopinavir and validation of HPLC method for the determination of related substances in lopinavir drug substance

Several related substances (RS4–RS10) were detected in lopinavir drug substance at levels ranging from 0.03% to 0.1% by employing gradient RP-HPLC. The related substances were identified by LC–MS analysis. These related substances were isolated and characterized by Mass, 1H NMR and FT-IR spectral data. The separation was achieved on a YMC Pack ODS-AQ (250 mm × 4.6 mm, 5 μm) column thermostated at 45 °C using 0.02 M KH2PO4 (pH 2.5): acetonitrile as a mobile phase in gradient elution mode. A PDA detector set at 210 nm was used for detection. The investigated validation elements showed the method has acceptable specificity, accuracy, linearity, precision, robustness and high sensitivity with detection limits and quantitation limits ranging from 0.028 μg/ml to 0.063 μg/ml and 0.084 μg/ml to 0.192 μg/ml respectively. The method can be used for routine quality control analysis and stability testing of lopinavir drug substance.