Development and Validation of a Rapid Solid-Phase Extraction: Ultrafast Liquid Chromatographic Method for the Estimation of Azithromycin and Its Major Related Substances in Human Plasma and Dosage Forms Using a Novel Polyfunctional Silyl Reagent-Bonded Core–Shell Column

• Published in: Chromatographia Vol. 82; no. 10; pp. 1489 - 1500
• Main Authors: Sahoo, Dibya Ranjan, Sahoo, Sunanda
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2019; Springer Nature B.V
• Subjects: Affinity; Analytical Chemistry; Binding; Blood plasma; Chemistry; Chemistry and Materials Science; Chromatography; Docking; Elution; Flow velocity; Hydrogen bonds; Impurities; Laboratory Medicine; Liquid chromatography; Original; Pharmacy; Plasma; Proteins; Proteomics; Reagents; Solid phases; Solvents; Azithromycin: validation; Azithromycin-related impurities; Ultrafast liquid chromatography; Novel polyfunctional silyl reagent core–shell columns; Solid-phase extraction
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1007/s10337-019-03768-z

In this study, a rapid reversed-phase liquid chromatography method for the determination of azithromycin and its related substances was developed and validated on a novel polyfunctional silyl reagent [hexanhexamethyloctadecyltetrasiloxane (HMODTS–C18)] bonded core–shell RP C-18 column (100 × 4.6 mm, 2.6 µm) as per ICH guidelines. A binary gradient elution programme with a mixture of solvent A (phosphate buffer, pH 8.9) and solvent B (ACN: MeOH, 3:1) as mobile phase, at a flow rate of 1.5 mL min
−1
and detection at 210 nm was finally optimized for the study. The validation results showed that the method to be specific, selective, highly sensible (0.004 mg mL
−1
), precise (% RSD ≤ 10), linear and accurate in a concentration range of 0.004–0.032 mg mL
−1
. Simple SPE method was performed for extraction and checked for repeatability. The result showed that the method is precise (%RSD < 2). The validated chromatographic method was applied to the solid-phase extracted samples of azithromycin and its four major related substances. Also, docking study was carried out using AutoDock Tools to find out the binding affinities, number of hydrogen bonds and residues involved in hydrogen bonds for azithromycin and its four major related impurities with the human plasma proteins. The results confirmed the applicability of the proposed method on the extracted human plasma samples. Finally, the study demonstrates that the impurities of azithromycin have strong binding affinities with the plasma proteins compared to that of azithromycin and half life of these impurities may be higher than azithromycin which can cause major health risk on repeated doses of azithromycin.