Simultaneous quantitative determination of insulin aspart and insulin degludec in human plasma using simple shoot LC method.

ABSTRACT

Combining short-acting and long-acting insulin analogs was a real challenge that was overcome by NovoNordisk through the co-formulation of insulin aspart and insulin degludec in single-dosage form. The proposed study provides a simple, short, and reliable HPLC method with diode array detection that 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 35°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 was applied using a 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 a 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%.