Development and validation of spectrophotometric methods for determination of phytomenadione in injection

Three simple and sensitive spectrophotometric methods have been developed for determinationof phytomenadione in injection. Method A is simple UV spectrophotometric method based ondetermination of phytomenadione in methanol at 249 nm. Linearity was obtained in theconcentration range of 2 – 14 ug/ml. Method B is first order derivative spectrophotometric method and involves estimation of phytomenadione in methanol using the first - order derivativetechnique at 259 nm as maxima and 276 nm as minima. Calibration curve was prepared byplotting the absorbance difference between maxima and minima versus concentration. Linearitywas obtained in the concentration range of 2-14 μg/ml. Method C is area under curve (AUC)method. The method involved calculation of integrated value of absorbance with respect to thewavelength between two selected wavelength 241 nm and 280 nm, respectively. Linearity wasobtained in concentration range of 2- 14 μg/ml. These methods were successfully applied topharmaceutical formulations because no interferences from injection excipients were found. Thesuitability of these methods for the quantitative determination of phytomenadione was proved byvalidation. The proposed methods were found to be simple and cost effective and can beemployed for the routine quality control application of phytomenadione in pharmaceuticalformulations. Keywords: Phytomenadione, Spectrophotometric, First order derivative, Area under curve,Injection, Validation.