Determination of Human Insulin in Pharmaceutical Preparation by Zero, First and Second Order Derivative Spectrophotometric Methods

Zero, first and second order derivative spectrophotometric methods were developed for the determination of insulin. The standard solutions and pharmaceutical sample were prepared in 0.01 M HCl. Absorbences of insulin were measured at 276 nm for the zero order by measuring height of peak from zero, at 264, 277 and 284 nm for the first order and at 277, 280, 283 and 289 nm for second order derivative spectrophotometric method by measuring peak to peak height. The type of solvent, the degree of deviation and range of wavelength were chosen in order to optimize the conditions. The linearity ranges were found to be 5.0-70 μg mL for the zero, first and second order derivative spectrophotometric methods. The detection and quantitation limits were found to be 2.0 μg mL & 3.0 μg mL for the zero order, 3.0 μg mL & 4.0 μg mL for first and second order derivative spectrophotometric methods, respectively. The developed zero, first and second order derivative spectrophotometric methods in this study are accurate, sensitive, precise, and reproducible and can be directly and easily applied to the pharmaceutical preparation. In addition, the results obtained from three derivative spectrophotometric methods were compared and no significant difference was found statistically.