Acceleration of analysis in quality control: Transfer of the classic UV/VIS spectrophotometric method for determination of lysozyme activity to a microtiter plate reader

In modern pharmaceutical quality control, accelerating analytical procedures while maintaining high accuracy is a key priority. This study focuses on optimizing analysis times by transferring classical UV/VIS spectrophotometric methods to a microplate reader platform. The model method chosen for this research was the determination of lysozyme enzymatic activity, conducted in accordance with the current Japanese Pharmacopoeia monograph. The underlying assay mechanism is based on spectrophotometrical measurement of the rate of absorbance decrease of a Micrococcus luteus bacterial suspension at approximately 450 nm. This decrease is a result of enzymatic hydrolysis of the cell wall and is directly proportional to the sample's activity relative to an official reference standard. The adapted method was verified and fully transferred from a standard cuvette-based spectrophotometer to a microplate reader format. This transfer enables simultaneous analysis of a large number of samples, resulting in significant time savings and higher throughput. Technical challenges encountered during the transition were successfully resolved by optimizing critical experimental parameters, including sample and reagent concentrations, volumes, reagent addition order, and reaction times, thereby ensuring strict result equivalence. The optimized method was successfully applied to three commercial products: Lysobact lozenges, Lysobact Junior oromucosal spray, and Lysoderm cream. Ultimately, verification of this model demonstrates that transferring to microplate readers enables high sample throughput while maintaining required accuracy and precision. This paper provides practical guidance for adapting classic UV/VIS spectrophotometric methods to modern technologies, improving efficiency in drug quality control.