Simultaneous quantitative determination of Insulin Aspart and Insulin Degludec in human plasma using simple shoot liquid chromatography method.

Combining short-acting and long-acting insulin analogs was a real challenge which was overcome by NovoNordisk through co-formulation of insulin aspart and insulin degludec in single dosage form. The proposed study provides a simple, short and reliable high-performance liquid chromatographic method with diode array detection which is developed and validated for the simultaneous determination of insulin aspart and insulin degludec in human plasma. The proposed method achieved good separation between the two analytes utilizing a C8-column at 350 C in a very short run time (6 min), with a simple, low cost and reliable extraction method through precipitation of plasma protein. Gradient elution is applied using mobile phase consisting of 0.1 M Sodium Sulfate (pH 3.4) and Acetonitrile. The method was validated according to EMA Guideline on Bioanalytical validation. The proposed method had linear range from 3.0 to 300 μg/mL for insulin aspart and from 3.5 to 300 μg/mL for insulin degludec. The intra- and inter-day precision of insulin aspart were 0.36 % - 3.33% and 1.59 % - 8.84 %, respectively, and accuracy was between-10.06 % and 3.09% The intra- and inter-day precision of insulin degludec were 0.29% - 1.93% and 0.89% - 5.14%, respectively, and accuracy was between -5.29% and 3.91%.