Premium
Development and validation of a rapid reversed‐phase HPLC method for the determination of insulin from nanoparticulate systems
Author(s) -
Sarmento Bruno,
Ribeiro António,
Veiga Francisco,
Ferreira Domingos
Publication year - 2006
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.616
Subject(s) - chemistry , chromatography , trifluoroacetic acid , high performance liquid chromatography , elution , gradient elution , relative standard deviation , acetonitrile , aqueous solution , detection limit , reversed phase chromatography , chitosan , phase (matter) , dosage form , biochemistry , organic chemistry
A reversed‐phase high‐performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin‐loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed‐phase (RP) C 18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1–100 µg/mL ( R 2 = 0.9996), specific with a good inter‐day and intra‐day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 µg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin‐loaded nanoparticles made from cross‐linked chitosan‐alginate. Copyright © 2006 John Wiley & Sons, Ltd.