Development and validation of a new robust RP-HPLC method for simultaneous quantitation of insulin and pramlintide in non-invasive and smart glucose-responsive microparticles

Jaber Emami , Maryam Haghighi, Mahboobeh Rostami, Mohsen Minaiyan, Mohsen Minaiyan


Background and purpose: Since insulin and pramlintide cooperate in glucose hemostasis, co-administration and quantitation of them in pharmaceutical preparations are imperative. A simple, rapid, sensitive, and isocratic RP-HPLC method was developed and validated for simultaneous quantitation of insulin and pramlintide in loading and in-vitro release studies of a glucose-responsive system to improve the control of hyperglycemic episodes in diabetic patients.

Experimental approach: The isocratic RP-HPLC separation was achieved on a C18 µ-Bondopak column (250 mm × 4.6 mm) using a mobile phase of water:acetonitrile:trifluoroacetic acid (65:35:0.1%) at a flow rate of 1 mL/min in an ambient temperature. Both proteins were detected using a UV detector at 214 nm. The method was validated for specificity, linearity, precision, accuracy, the limit of detection, the limit of quantification, and robustness.

Findings/Results: Linearity was obtained in the concentration range of 30 to 360 μg/mL for insulin and 1.5 to 12 μg/mL for pramlintide. The results were validated statistically and recovery studies confirmed the great accuracy and precision of the proposed method. The robustness of the method was also confirmed through small changes in pH, mobile phase composition, and flow rate.

Conclusion and implications: The method was found to be simple, specific, precise, and reproducible. It was applied for the determination of loading capacity, entrapment efficiency, and in-vitro release studies of insulin and pramlintide in a smart glucose-responsive microparticle. Co-delivery of insulin and pramlintide could be a new intervention in diabetes management and concurrent quantitation of these two proteins is, therefore, essential.


Diabetes; Insulin; Pramlintide; RP-HPLC; Smart-glucose responsive microparticles.


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