A New Short Validated U-HPLC Method for the Determination of Recombinant Human Insulin in Microspheres

Background: The objective of present research work was to develop and validate rapid UHPLC method with short run time for the detection and quantification of recombinant human insulin content in microsphere formulations. Validation emphasizes mainly on studying the effects of the flow rate, wavelengths, column temperature, pH and organic content of the mobile phase and of presence of other additives on the method's accuracy. Methods: Chromatographic separation was achieved using YMC Triart C-18 column and the mobile phase was composed of the mixture (aqueous solution of 0.2M anhydrous sodium sulphate, pH 2.3 and acetonitrile) in a ratio 73:27 (%v/v). The column temperature and flow rate was 40ºC and 0.613 mL/min respectively whereas, UV detection was performed at 214 nm. All the method procedures were validated according to international conference on harmonization (ICH) guidelines. The retention time of recombinant human insulin was about 3.0 min with very short run time of just 5 min. Fluctuations in analytical conditions or presence of additives and impurities or insulin related analogs did not show any significant effect on the specificity and accuracy of the method. Results: The assay is reproducible with acceptable %RSD value (0.87%). Linearity was confirmed by correlation coefficient of 0.9999 over the range of 26.84-81.23 μg/mL. The accuracy of the method and % recovery of insulin content were found to be in between 98.02 and 99.71% while RSD ranged from 0.01 to 0.88%. The specificity of the method proved that no interferences occurred with RT of insulin peak. Conclusion: This new short validated method can be applied during development studies in research laboratories for high throughput testing as well as for quality control procedures of lot release testing, in-process quality control and finished product analysis.