Turbidimetric and HPLC assays for the determination of formulated lysozymeactivity

In several studies lysozyme has been employed as a model protein to investi gate the effects of formulation factors upon biological activity. The aim o f this work was to develop and validate an HPLC technique to assay lysozyme and to compare the results with biological activity determined from a vali dated turbidimetric assay. The turbidimetric assay was based upon the lytic action of lysozyme on Micrococcus lysodeikticus cells, whilst the reverse- phase HPLC assay employed an acetonitrile gradient in 0.1 % trifluoroacetic acid. The limits of detection and quantification were 3.84 and 6.24 mug mL (-1) for HPLC assay, whilst the corresponding Values for turbidimetric assa y were 1.94 and 3.86 mug mL(-1). The methods were used to monitor the loss of enzyme activity after heating. Lysozyme concentrations determined from H PLC peak height were found to correlate (r(2) = 0.9963) with those obtained from turbidimetric assay.