Protein-based APIs represent a big group of modern therapeutics. Their characterization involves complex analytical protocols which require special methods, especially in the case when the protein drug is included into tablet dosage forms. Although the fluorescence polarization assay (FPA) is not currently regulated by many national Pharmacopeias, it represents a promising approach for protein drug standardization, considering their rapid, sensitive, and automatable detection suitable for high-throughput analysis and real-time quality control. To evaluate the applicability of FPA for the analysis of protein drugs in tablets, the quantifying of lysozyme in tablet dosage forms was studied by this method with the use of a fluorescently labeled synthetic chitooligosaccharide tracer. It was shown that this approach overcomes the limitations of the conventional turbidimetric assay of lysozyme determination, which is labor-intensive and relies on unstable reagents. Measurements were performed with both portable and stationary fluorescence polarization readers. Commercial tablets from five manufacturers containing lysozyme (20 mg) and pyridoxine hydrochloride (10 mg) together with other excipients were analyzed. The FPIA method showed a linear range of 5.0–70 µg/mL, with specificity confirmed by the absence of interference from excipients. Accuracy, evaluated by standard addition (10–20 mg), yielded recoveries of 100.2–106.0%. Placebo spiked with lysozyme at 80–120% of nominal content demonstrated recoveries of 98.0–100.1%, with RSD (n = 6) not exceeding 13.7%, indicating good precision. The developed method enables reliable lysozyme quantification in tablets, offering speed, simplicity, and robustness, and shows its suitability for the routine quality control of protein-containing dosage forms including the enzyme ones.

Introduction. Acetochlor is one of the most common herbicides capable of accumulating in medicinal plant raw materials and posing a health risk. A method for acetochlor determination was developed using fluorescence polarization immunoassay (FPIA), a homogeneous and specific technique employed for analysis of low-molecular-weight toxicants. Aim. To develop and validate a fluorescence polarization immunoassay for quantitative analysis of the herbicide acetochlor in medicinal plant material, exemplified by corn silk ( Zea mays L.). Materials and methods. For the development of the method, polyclonal rabbit antibodies against acetochlor and a tracer consisting of an acetochlor derivative conjugated with ethylenediamine fluorescein (AMPA-EDF) were used. The analysis was performed by fluorescence polarization using a portable Sentry-200 fluorimeter (Ellie LLC, USA). Data processing was carried out with SigmaPlot 11 software package (Systat Software Inc., USA). The study was performed on samples of corn silk medicinal plant material purchased from a local pharmacy and an online store. Method validation included the following parameters: limit of detection, limit of quantification, analytical range, precision, accuracy, and specificity. Results and discussion. A method for the quantitative determination of the pesticide acetochlor in the herbal medicinal raw material corn silk ( Zea mays L.) has been developed, optimized, and validated using fluorescence polarization immunoassay (FPIA). The determination range was from 0.024 to 1.56 mg/kg. The specificity, precision, and accuracy of the developed method were confirmed. Conclusion. The fluorescence polarization immunoassay (FPIA) method was applied for the quantitative determination of the pesticide acetochlor in herbal medicinal raw material, using corn silk as an example.

Hen egg white lysozyme (HEWL) is a well-characterized antibacterial enzyme widely used in research and industry. However, little is known about the functional properties of lysozyme from other bird species, such as pheasants. This study aimed to compare the muramidase and chitinase activities of HEWL with the lysozyme from pheasant egg white (PEWL). Egg white samples were obtained from ten hens and ten pheasants. Lysozyme activity was assessed by two methods: a turbidimetric assay using Micrococcus luteus cell suspension to measure bacteriolytic (muramidase) activity and a fluorescence polarization assay (FPA) using FITC-labeled tri- and chitopentaoside substrates to measure the chitinase activity. The observed dissociation rate constant (k_obs) was derived from the time-dependent FP signal. Turbidimetric measurements showed that PEWL possessed bacteriolytic activity approximately 30% lower than that of HEWL under the same conditions. However, FPA revealed a striking functional difference: whereas HEWL only bound the chitotrioside substrate without hydrolyzing it, PEWL was able to hydrolyze chitotriose. Both enzymes readily hydrolyzed the chitopentaoside substrate. The hydrolysis rate of the chitopentaoside was 50–70 times higher than that of the chitotrioside for PEWL. Sequence comparison identified six amino acid substitutions in PEWL (Phe3 → Tyr, His15 → Leu, Gln41 → His, Asn77 → His, Gln121 → Asn, Ile124 → Thr) relative to HEWL, which likely alter the conformation of subsites A–C and enable productive binding of the shorter oligosaccharide. Thus, despite high sequence similarity, pheasant lysozyme exhibits a unique functional property—the ability to hydrolyze chitotriose into N-acetylglucosamine. These findings expand functions of avian lysozymes and highlight the potential of alternative bird species as sources of enzymes with novel properties.