CONFERENCE PROCEEDING
Application of the method of fluorescence polarization analysis for lysozyme assay in tablet dosage form
1
Institute of Pharmacy, Federal State Autonomus Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of Ministry of Health of the Russian Federation (Sechenovskiy University), Moscow, Russia
2
Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia
3
N.D. Zelinsky Institute of Organic Chemistry RAS, Moscow, Russia
Publication date: 2026-07-30
Public Health Toxicol 2026;6(Supplement 1):A9
KEYWORDS
ABSTRACT
Introduction:
Lysozyme, a natural enzyme with muramidase and chitinase activity, is used medicinally for its antimicrobial properties. While pharmacopoeial methods rely on turbidimetric Micrococcus lysodeikticus assays—which are labor-intensive, time-consuming, and require unstable biological reagents—this study developed a fluorescence polarization assay (FPA) based on lysozyme’s interaction with a fluorescently-labeled peptidoglycan trisaccharide. The aim was to validate this FPA for rapid, reliable quantification of lysozyme in tablets.
Methods:
Egg white lysozyme (≥15,000 U/mg; Sisco Research Laboratories) and a fluorescently-labeled chitin oligosaccharide tracer (Mukhametova et al.) were used. Analyses employed a Sentry 200 fluorescence polarization analyzer (Ellie LLC). Commercially available tablets (20 mg lysozyme + 10 mg pyridoxine; six manufacturers) were analyzed. Validation assessed analytical range, specificity, accuracy, and precision.
Results:
Lysozyme quantification in tablets employed parallel analysis of 200.0, 400.0, and 600.0 mg samples with mean calculation, essential for enzymatic activity-based methods. This approach evaluated matrix effects, verified dilution-independent activity consistency, and optimized working dilution to minimize interferences. Parallel testing demonstrated convergent results across all dilutions, enabling reliable analysis across the entire validated mass range. The validated method demonstrated analytical range 150-650 μg/mL and specificity with no significant cross-reactivity to excipients. The accuracy of the method was confirmed via a standard addition approach by spiking 10 mg and 20 mg of lysozyme reference standard into homogenized tablet powder, demonstrating satisfactory recovery rates of 100.2–106.0%. Precision, assessed via six replicates, demonstrated an RSD ≤ 13.7%.
Conclusion:
A validated method for lysozyme quantification in tablets was developed for quality control, offering rapid analysis, reduced labor, and elimination of live microbiological reagents.
Lysozyme, a natural enzyme with muramidase and chitinase activity, is used medicinally for its antimicrobial properties. While pharmacopoeial methods rely on turbidimetric Micrococcus lysodeikticus assays—which are labor-intensive, time-consuming, and require unstable biological reagents—this study developed a fluorescence polarization assay (FPA) based on lysozyme’s interaction with a fluorescently-labeled peptidoglycan trisaccharide. The aim was to validate this FPA for rapid, reliable quantification of lysozyme in tablets.
Methods:
Egg white lysozyme (≥15,000 U/mg; Sisco Research Laboratories) and a fluorescently-labeled chitin oligosaccharide tracer (Mukhametova et al.) were used. Analyses employed a Sentry 200 fluorescence polarization analyzer (Ellie LLC). Commercially available tablets (20 mg lysozyme + 10 mg pyridoxine; six manufacturers) were analyzed. Validation assessed analytical range, specificity, accuracy, and precision.
Results:
Lysozyme quantification in tablets employed parallel analysis of 200.0, 400.0, and 600.0 mg samples with mean calculation, essential for enzymatic activity-based methods. This approach evaluated matrix effects, verified dilution-independent activity consistency, and optimized working dilution to minimize interferences. Parallel testing demonstrated convergent results across all dilutions, enabling reliable analysis across the entire validated mass range. The validated method demonstrated analytical range 150-650 μg/mL and specificity with no significant cross-reactivity to excipients. The accuracy of the method was confirmed via a standard addition approach by spiking 10 mg and 20 mg of lysozyme reference standard into homogenized tablet powder, demonstrating satisfactory recovery rates of 100.2–106.0%. Precision, assessed via six replicates, demonstrated an RSD ≤ 13.7%.
Conclusion:
A validated method for lysozyme quantification in tablets was developed for quality control, offering rapid analysis, reduced labor, and elimination of live microbiological reagents.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest in the publication of this article. The authors have no conflicts of interest to report in this work.
Abstract was not submitted elsewhere and published here firstly.
FUNDING
This work was supported by the Russian Science Foundation (grant no. 19-73-30017-P).
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| eISSN: | 2732-8929 |
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