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

Abstract

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.

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

Fig. 1

Chemical structures of (A) insulin and (B) pramlintide

Fig. 2

Chromatogram of (A) insulin and pramlintide prepared in the mobile phase, and (B) mobile phase injected as the control. Insulin concentration: 195 µg/mL, pramlintide concentration: 6.5 µg/mL.

Fig. 3

Chromatogram of unloaded proteins in the supernatant of (A) smart glucose-responsive microparticles containing insulin at 101 µg/mL and pramlintide at 2.9 µg/mL, and (B) plain smart-glucose responsive microparticles in loading medium.

Fig. 4

Chromatogram of released proteins from (A) smart glucose-responsive microparticles containing insulin and pramlintide and (B) plain smart glucose-responsive microparticles in release medium (released insulin concentration: 241 µg/mL; pramlintide concentration: 9.3 µg/mL).

Fig. 5

Calibration curves of (A, C, E) insulin and (B, D, F) pramlintide in the mobile phase, loading, and release media. Each point represents means ± SD, n = 3. Due to small variations amongst experiments, in many data points, SD bars are not visible.

Fig. 6

Chromatograms of different concentrations of insulin and pramlintide in (A) mobile phase, (B) loading medium, and (C) release medium. In each set of chromatograms A₁, B₁, and C₁ represent low concentration, insulin at 30 µg/mL and pramlintide at 1.2 µg/mL; A₂, B₂, and C₂ represent medium concentration, insulin at 150 µg/mL and pramlintide at 5 µg/mL; and A₃, B₃, and C₃ show the highest concentration, insulin at 360 µg/mL and pramlintide at 12 µg/mL.

Fig. 7

Release profiles of insulin and pramlintide liberated from smart glucose-responsive microparticles. Each point represents means ± SD, n = 3.