Development and validation of UHPLC-DAD method for the determination of cholesteryl-hexahydrophthaloyl-5-fluorouracil in lipid nanoemulsion

An ultra HPLC-diode array detector (UHPLC-DAD) method was developed and validated for rapid determination of cholesteryl-hexahydrophthaloyl-5-fluorouracil (CH5F) conjugate in standard drug, lipid nanoemulsion and dissolution samples. The chromatographic identification of this conjugate was achieved on Hypersil GOLD 50 x 2.1 mm reversed phase C-18 column having a 1.9 mu m packing as a stationary phase using methanol-water (80: 20, v/v) as a mobile phase, at a flow rate of 0.4 mL/min with DAD detection at 276 nm. The proposed UHPLC-DAD method is linear in the concentration range of 1-50 mu g/mL with correlation coefficient of 0.998. The proposed method is precise, accurate, robust, sensitive and specific for analysis of the conjugate. High assay value (98.7%) of CH5F conjugate in lipid nanoemulsion was obtained by the proposed method. Forced degradation studies indicated that conjugate was sufficiently stable under oxidative stress conditions degraded under acid, base and thermal stress conditions. The proposed UHPLC-DAD method successfully and resolved drug conjugate peak in the presence of its degradation products which established stability and indicating property of the method. Results of the present study indicated that the proposed UHPLC-DAD method can be successfully used for routine determination of CH5F conjugate in standard drug and pharmaceutical formulations.