A Validated RP-HPLC Method for the Estimation of Related Substances of Gemcitabine in Gemcitabine Injection 38 mg/mL

Abstract

A novel, simple, sensitive and stability-indicating high-performance liquid chromatography method was developed and validated for the related substances of Gemcitabine in Gemcitabine Injection 38mg/vial. Reversed-phase chromatography was performed on Agilent 1100 series with Software Empower-2  and Agilent 1100 series with PDA 1100  series Software Empower-2 photodiode array detector using ZorbaxRx C8 (250 mm × 4.6 mm, 5 µm particle size) column with pH 3.0 (adjusted with Ortho Phosphoric acid) of monobasic sodium phosphate buffer as mobile phase-A and Methanol as Mobilephase-B at a flow rate of 1.2 mL/min. Gradient profile at Initial: 97-3, 8 minutes: 97-3, 13 minutes: 50-50, 20 minutes: 50-50, 25minutes: 97-3, 35minutes: 97-3  and with UV detection at 275 nm.  Linearity was observed in the concentration range of Cytosin 0.04-3.0 µg/mL (R2 = 0.999), Alpha Anomer impurity 0.10–2.91 µg/mL (R2 = 0.999), the concentration range of Gemcitabine 0.15-5.5 µg/mL (R2 = 0.999), and the concentration range of Beta Uridine Impurity 0.05–72.5µg/mL (R2 = 0.999),). The  limit of detection (LOD) AND limit of Quantitation (LOQ) were found to be Cytosin impurity 0.02&0.04 µg/mL, Alpha Anomer impurity 0.06&0.15µg/mL, Gemcitabine 0.06&0.17 µg/mL and Beta Uridine Impurity 0.02&0.06 µg/mL, respectively. The method was validated as per ICH guidelines. The RSD for intra-day (0.1-1.3) and inter-day (0.5-1.1) precision were found to be less than 10.0 %. The percentage recovery was in good agreement with the labelled amount in the pharmaceutical formulations and the method is simple, specific, precise and accurate for the determination of related substances Gemcitabine in pharmaceutical formulations.