Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
Spectrochim Acta A Mol Biomol Spectrosc. 2025 Jan 15;325:125079. doi: 10.1016/j.saa.2024.125079. Epub 2024 Sep 1.
Tulathromycin is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The aim of this study was the development of two innovative microwell spectrometric (photometric and fluorometric) assays for determination of tulathromycin in its pharmaceutical formulations. To achieve this goal, 4-fluoro-7-nitrobenzofurazan was investigated as a dual-function chromogenic and fluorogenic probe for tulathromycin. The reaction between tulathromycin and 4-fluoro-7-nitrobenzofurazan proceeded smoothly in an alkaline medium, resulting in the formation of a colored and fluorescent product. The product displayed a maximum light absorption at 475 nm and emitted fluorescence at 545 nm when excited at 475 nm. Extensive investigations were conducted to optimize the factors affecting the tulathromycin /4-fluoro-7-nitrobenzofurazan reaction, and the optimal conditions were established. Using these optimized conditions, both microwell-based photometric and fluorometric assays were developed. The calibration curves relating the absorbance and fluorescence intensities of the reaction product with the corresponding tulathromycin concentrations were generated. The absorbance-concentration relationship was found to be linear within a tulathromycin concentration range of 10-400 µg mL, with a limit of quantitation of 6.2 µg mL. On the other hand, the fluorescence-concentration relationship was linear within a concentration range of 0.04-1.2 µg mL, with a limit of quantitation of 0.06 µg mL. Rigorous validations of both assays' procedures were performed, and both assays were successfully employed for the analysis of tulathromycin-containing pharmaceutical formulations (injections) with satisfactory accuracy and precision. The ecologically friendly assessment of both assays demonstrated their compliance with the principles of green analytical chemistry approaches. Moreover, the proposed microwell-based assays enabled the simultaneous analysis of multiple samples using small volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of 4-fluoro-7-nitrobenzofurazan as a probe with dual functionality for the microwell-based photometric and fluorometric analysis of tulathromycin. The developed assays serve as valuable analytical tools for ensuring the quality of tulathromycin 's pharmaceutical formulations.
替米考星是一种三脒类抗菌药物，已在欧盟和美国获得批准，用于治疗和预防牛的呼吸道疾病。本研究的目的是开发两种创新的微孔板分光光度（比色和荧光）测定法，用于测定其药物制剂中的替米考星。为了实现这一目标，研究了 4-氟-7-硝基苯并呋咱作为替米考星的双功能显色和荧光探针。替米考星与 4-氟-7-硝基苯并呋咱在碱性介质中反应顺利，形成有色和荧光产物。当用 475nm 激发时，产物在 475nm 处显示最大光吸收，在 545nm 处发射荧光。进行了广泛的研究以优化影响替米考星/4-氟-7-硝基苯并呋咱反应的因素，并确定了最佳条件。利用这些优化条件，建立了基于微孔板的比色和荧光测定法。生成了与相应替米考星浓度相关的反应产物的吸光度和荧光强度的校准曲线。发现吸光度-浓度关系在替米考星浓度范围为 10-400μg mL 内呈线性，定量限为 6.2μg mL。另一方面，荧光-浓度关系在浓度范围为 0.04-1.2μg mL 内呈线性，定量限为 0.06μg mL。对两种测定方法的程序进行了严格的验证，两种测定方法均成功地用于分析含有替米考星的药物制剂（注射液），具有令人满意的准确性和精密度。对两种测定方法的生态友好性评估表明，它们符合绿色分析化学方法的原则。此外，所提出的基于微孔板的测定法能够使用小体积同时分析多个样品，实现高通量分析。总之，本研究首次评估了 4-氟-7-硝基苯并呋咱作为一种具有双重功能的探针，用于替米考星的微孔板比色和荧光分析。所开发的测定法是确保替米考星药物制剂质量的有价值的分析工具。
