Development and validation of stability indicating HPLC methods for related substances and assay analyses of amoxicillin and potassium clavulanate mixtures

• Published in: Journal of pharmaceutical and biomedical analysis 2017-03, Vol.136, p.1-9
• Main Authors: Bellur Atici, Esen, Yazar, Yücel, Ağtaş, Çağan, Ridvanoğlu, Nurten, Karlığa, Bekir
• Format: Article
• Language: English
• Subjects: Amoxicillin; Amoxicillin-Potassium Clavulanate Combination - analysis; Amoxicillin-Potassium Clavulanate Combination - chemistry; Amoxicillin-Potassium Clavulanate Combination - standards; Anti-Bacterial Agents - analysis; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - standards; Assay; Chromatography, High Pressure Liquid - methods; Clavulanic acid; Drug Contamination - prevention & control; Drug Stability; Molecular Structure; Related substances; Reproducibility of Results; Sensitivity and Specificity; Stress-testing; Validation
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2016.12.032

[Display omitted] •New stability-indicating HPLC methods were developed for amox-clav mixtures.•18 amox and 6 clav impurities were successfully separated by the developed method.•Both assay and related substances methods were validated according to ICH.•Process related amox imp. P was synthesized and characterized by NMR and LC/MS.•Stress-testing and stability studies of amox-clav mixtures were carried out. Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the β-lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic methods were reported for their quantification in mixtures. In the present work, single HPLC method for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and validated according to international conference on harmonization (ICH) guidelines. Eighteen amoxicillin and six potassium clavulanate impurities were successfully separated from each other by using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm×4.6mm, 3μm) column with 50μL injection volumes at a wavelength of 215nm. Commercially unavailable impurities were formed by degradation of amoxicillin and potassium clavulanate, identified by LC–MS studies and used during analytical method development and validation studies. Also, process related amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing and stability studies of amox/clav mixtures was carried out under specified conditions according to ICH and analyzed by using validated stability-indicating assay and related substances methods.