Analysis of lysozyme in human saliva by CE-UV: A new simple, fast, and reliable method

Many studies have shown a correlation between salivary lysozyme levels and the presence of various diseases or stress. Here, we developed a new, simple method for the analysis of lysozyme in human saliva by capillary electrophoresis (CE) and UV detection that has potential application in clinical settings. The optimized method used 1 M formic acid +10 % (v/v) isopropyl alcohol as a background electrolyte, transient isotachophoresis as a preconcentration method, deactivation of the glass insert surface by carbonic anhydrase as a blocking agent to eliminate non-specific protein adsorption, and only simple dilution as a sample pretreatment. The validation showed high linearity (r² = 0.9997), inter- and intra-day accuracy (-2.03 to +5.36 %) and precision (<5 % RSD), and limit of detection (0.5 μg/mL). The stability of lysozyme in human saliva stored at -80 °C for 7 days using protease inhibitors and the variations of lysozyme excretion during the day and over 3 consecutive days were also investigated. The optimized and validated method was applied for the determination of salivary lysozyme levels in 25 healthy volunteers and the results showed the mean salivary lysozyme concentration at 14.3 ± 10.5 μg/mL. To the best of our knowledge, this is the first CE-UV method for human salivary lysozyme determination. This method meets the criteria of simplicity (minimum steps in sample pretreatment and analysis), speed (the entire process from saliva collection to result takes less than 45 min), reliability (highly satisfactory validation data), and suitability for application to real-world samples.