Optimization and validation of a chromatographic method for quantification of lysozyme in jenny milk

Jenny milk has recently stimulated scientific interest for its attractive nutritional characteristics, particularly for geriatric and paediatric patients. Moreover, it has a high concentration of lysozyme, whose antibacterial properties are well recognized. In literature, a wide range of lysozyme concentrations in jenny milk has been reported (from 1.0 g[middot]l super(-1) to 4.0 g[middot]l super(-1)). In this study, a step-by-step optimized protocol for jenny milk lysozyme extraction and quantification by high pressure liquid chromatography (HPLC) was developed. Moreover, a comparison between ultraviolet (UV; at 227 nm and 280 nm) and fluorescence detection was performed. All developed HPLC methods for analysis of lysozyme were validated according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines (ICH Q2(R1), 2005) for linearity, accuracy, sensitivity and precision. The protocol here presented is easy and quick to perform, with the UV detection at 280 nm providing a valid alternative to traditional fluorescence method for quantification of lysozyme in jenny milk.