This paper describes the development and validation of a most economical and sensitive isocratic stability indicating
HPLC method for the assay of recombinant human insulin (RHI) from yeast origin (Hansenula polymorpha). The
method employs a Thermo Biobasic, C-18 (250 x 4.6 mm, 300 A°, 5 µm) column with a mobile phase of acetonitrile - potassium
dihydrogen phosphate ( pH 2.5; 50 mM) (35:65, v/v) and UV detection at 242 nm. Factorial design was used to
facilitate method development. Buffer pH and concentration of acetonitrile were considered as the independent variable to
study capacity factor of RHI. Sixteen experiments were undertaken, and a quadratic model was derived for the capacity
factor of RHI. The method produced well defined, sharp peaks having lower tailing factor (1.114). A linear response with
a correlation coefficient (r2) of 0.999 was observed over the range of 0.025 - 2.0 IU mL-1. Developed method was employed
to analyze RHI samples from forced degradation and stability study. Degradation of RHI under different ICH prescribed
stress conditions was carried out. Four batches of RHI formulation were exposed to different conditions of temperature
and humidity for forty-five days. Degradation of RHI during stability study followed first-order kinetics. Data obtained
from degradation kinetics were employed to find out the rate constant; time left for 50% potency, and time left for
90% potency. Rate constants obtained from different conditions were employed to plot the Arrhenius plot.
Keywords: Recombinant human insulin, HPLC, Stability indicating, Method validation, Degradation kinetics, Stability study.
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