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
- 24 Downloads
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.
KeywordsAzithromycin: validation Azithromycin-related impurities Novel polyfunctional silyl reagent core–shell columns Solid-phase extraction Ultrafast liquid chromatography
The authors are highly grateful to ImpScience Pvt. Ltd, Hyderabad, Telangana, India, for providing the necessary facilities during the research work.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 2.The United States Pharmacopoeia 36-NF 31 (2013) Azithromycin Tablets, United States Pharmacopeia Convention, Rockville, pp 2580–2582Google Scholar
- 4.Wildfeuer A, Laufen H, Zimmermann T (1994) Distribution of orally administered azithromycin in various blood compartments. Int J Clin Pharm Ther 32:356–360Google Scholar
- 5.Tsukamoto T, Nagae N, Silver S (2013) Usability of a core shell column using high performance liquid chromatograph for a routine analysisGoogle Scholar
- 6.Nagae N, Tsukamoto T, Grahn A, Jutvik R, Gaitonde VD (2011) A novel bonding technique using a polyfunctional silyl-reagent for reversed-phase liquid chromatography—a NEW APPROCHGoogle Scholar
- 7.The United States Pharmacopoeia 34-NF 29 (2011) Chapter < 621 > –chromatography. United States Pharmacopeia Convention, RockvilleGoogle Scholar
- 8.Neue UD, McCabe D, Ramesh V, Pappa H, DeMuth J (2009) Transfer of HPLC procedures to suitable columns of reduced dimensions and particle sizes, The United States pharmacopoeia. Pharmacop Forum 35:1622Google Scholar
- 9.European Pharmacopoeia 7.0 (2010) Chromatographic Separation Techniques 2.2.46: 4412Google Scholar
- 10.British Pharmacopoeia (2009) Adjustment to chromatographic conditions, pp 9Google Scholar
- 11.Pharmacopoeia Indian (2007) Liquid Chromatogr 2.4.14:128Google Scholar
- 13.Nagae N, Enami T, Doshi S (2002) The retention behavior of reversed-phase HPLC columns with 100% aqueous mobile phase. LC/GC 20:964–972Google Scholar
- 15.The United States Pharmacopoeia 34-NF 29 monographs (2011) United States pharmacopeia convention. Rockville 1998–2003:2006–2008Google Scholar
- 16.ICH Guidelines (2005) Validation of analytical procedures: text and methodology Q2(R1). In: International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, pp 1–13Google Scholar
- 17.Babu VVS, Sudhakar V, Murthy TEGK (2014) J Chromatogr Sep Tech 5:209Google Scholar
- 23.Trott O, Olson A (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comp Chem 31:455–461Google Scholar