## 1 Introduction  
The stability of an insulin solution can be significantly reduced by a number of chemical changes to its primary structure which result in insulin derivatives with modified secondary and quaternary structures resulting in denaturation, aggregation, and precipitation. This leads to drastic reductions in the hormone's biological functionality posing serious problems for its use in drug delivery systems [1; 2; 3; 4; 5; 6]. During storage and use, insulin deteriorates via two main chemical reactions: deamidation from hydrolysis and polymerisation from the formation of intermolecular covalent bonds with other insulin molecules to form higher molecular weight transformation products [6; 7]. Deamidation is a reaction in which the side chain group in glutaminyl or asparagineyl residues is hydrolysed to form a free carboxylic acid. The six residues in insulin which pose as potential deamidation sites are GlnA5, GlnA5, AsnA18, AsnA21, AsnB3 and GlnB4. The asparagine (Asn) residues are more prone to deamidation than glutamine (Gln) residues. Deamidation products essentially retain native activity and are not associated with adverse immunogenicity [6]. In acidic solutions (pH <2) direct hydrolysis of the side chain amide leads to deamidation at AsnA21 and forms A21 desamido insulin [8]. Thisalters the charge and hydrophilic/hydrophobic properties of insulin which are the key forces controlling its tertiary structure and responsible for its biological functionality. However, in neutral solutions, deamidation primarily occurs at residue AsnB3 but at a reduced rate. Deamidation of AsnB3 and hydrolysis of the peptide bond between residues A8 and A9 contribute to formation of a mixture of iso-aspartate (isoAsp) and aspartate (Asp) derivatives (B3 desamido insulin) [9; 10]. Reversed phase high performance liquid chromatography (RP-HPLC) has been widely used to develop and validate simple specific methods with short run times for the determination of insulin in biological environments [11; 12; 13; 14]. The British Pharmacopoeia official monographs [15] highlight a HPLC method for the assay of human insulin and A21 desamido insulin utilising a gradient method with a run time of 50 min, whilst the United States Pharmacopoeia [16] describes a method for similar analysis at a longer run time of 90 min. A RP-HPLC method described by [PERSON] et al. [17] for the analysis and characterisation of a rapid acting insulin (Novo Nordisk(r)) and its degradation products in pharmaceutical preparations was able to detect A21 desamido insulin when formulations were stored at 60 \({}^{\circ}\)C with shaking. However, B3 degradants were detected for samples stored at 50 \({}^{\circ}\)C without shaking. The validation studies carried in this study were also performed using pure crystalline insulin and not the pharmaceutical preparations where A21 and B3 degradants were detected. However, as both degradation products are formed as a function of the pH environment a single HPLC method which has been able to capture both degradation products remains elusive. An isocratic HPLC method reported by [PERSON] et al. [18] detected A21 desamido insulin prepared by storing human insulin in 0.01 M HCl at 40 \({}^{\circ}\)C for 48 h with the peak appearing close to the human insulin peak but not B3 degradants. The aim of this work was to develop and validate two RP-HPLC methods at acidic and neutral pH for the quantification of insulin and A21 and B3 desamido insulin degradants in accordance with United States Food and Drug Administration (FDA) -approved methodology [19]. Both RP-HPLC methods were subjected to an assessment of selectivity, system suitability, linearity, accuracy, precision, sensitivity and stability for the quantification of insulin and A21 and B3 desamido insulin degradants, respectively, using Humulin(r) R (Hum R) and not a pure crystalline insulin as with [PERSON] et al. [17].