Copyright © 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 898–903 (2006) 898 B. Sarmento et al. ORIGINAL RESEARCH ORIGINAL RESEARCH Copyright © 2006 John Wiley & Sons, Ltd. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 20: 898–903 (2006) Published online 3 January 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bmc.616 Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems Bruno Sarmento, 1 * António Ribeiro, 2 Francisco Veiga 3 and Domingos Ferreira 1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal 2 Department of Pharmaceutical Technology, Instituto Superior de Ciências da Saúde-Norte, Gandra, Portugal 3 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Received 9 August 2005; revised 31 October 2005; accepted 1 November 2005 ABSTRACT: 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 un- der 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. KEYWORDS: alginate; chitosan; insulin; method validation; nanoparticles; RP-HPLC production is not enough to ensure daily needs of this hormone. It is a well characterized protein with a molecular weight of 5.8 kDa and composed of two peptide chains, referred to as the A-chain of 21 amino acid residues and the B-chain of 30 amino acid residues. Two disulfide bridges link these chains together and another intrapeptide disulfide bond exists in the A-chain (Brange et al., 1997). The desire of a more convenient and socially com- patible route of insulin administration other than sub- cutaneous injection has originated several approaches to attempt its oral delivery. The association of insulin with colloidal drug carriers which might be able to pro- tect proteins inside the intestinal tract and facilitate its transport from the gut lumen to the blood com- partment has been suggested (Aboubakar et al., 2000; Michel et al., 1991). Several pre-formulation studies and in vitro and in vivo assays have been performed to- wards the optimization of insulin oral delivery systems (Hari et al., 1996; Marschutz and Bernkop-Schnurch, 2000; Onal and Zihnioglu, 2002; Takenaga et al., 2002), requiring appropriated and validated methods to assess truthful profiles of insulin release from delivery systems as well as its pharmacokinetic behaviour. However, it has been reported that not only analytical techniques used for the development of pharmaceuticals must be INTRODUCTION Reversed-phase HPLC, a technique used to separate proteins based on hydrophobicity, is probably the most used analytical method for separation and determina- tion of peptides and proteins in an extensive range of applications (Aguilar, 2004). The method is powerful and sensitive, being an accurate way to measure insulin in both artificial and biological environments (Khaksa et al., 1998; Moslemi et al., 2003; Oliva et al., 2000). Also, official monographs, namely the US and European Pharmacopoeia, present HPLC methods for insulin determination. However, these methods usually require long run times and use buffers as mobile phases that are often responsible for contamination of HPLC systems due to salt precipitations. Insulin is a protein administered subcutaneously to humans for the treatment of type I diabetes mellitus to control glucose homeostasis when pancreatic β -cells *Correspondence to: B. Sarmento, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal. E-mail: bruno.sarmento@ff.up.pt Abbreviations used: ACN, acetonitrile; AE, association efficiency; LC, loading capacity; TFA, trifluoroacetic acid. Contract/grant sponsor: Fundação para a Ciência e Tecnologia.