METHOD DEVELOPMENT AND VALIDATION OF ANTIBIOTICS BY RP-HPLC WITH EMPHASIS ON STABILITY STUDIES: A COMPREHENSIVE REVIEW

Md. Saniya, Sreelatha Gangu*, Mallani Sirisha, Sarvepalli Gnana Samanvitha, Shaik Naziya Fathima, Arunabha Mallik

ABSTRACT

Antibiotics remain a cornerstone of global healthcare, yet increasing antimicrobial resistance and strict qualitycontrol
requirements necessitate highly accurate and robust analytical methods for their quantification. Reversedphase
high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique for the
routine and stability-indicating analysis of antibiotic drug substances and formulations. This review provides a
comprehensive overview of RP-HPLC method development strategies, validation requirements and forced
degradation studies specific to antibiotics. It examines a wide range of chromatographic conditions applied to β-
lactams, macrolides, fluoroquinolones, cephalosporins, tetracyclines and aminoglycosides. A systematic literature
review summarizes major analytical methods reported in the last two decades, highlighting chromatographic
challenges, degradation pathways, impurity profiling and method robustness considerations. Emerging analytical
trends such as green chromatography, Quality by Design (QbD) and hyphenated techniques (PDA/MS) are also
discussed. The review emphasizes the need for stability-indicating analytical methods to comply with ICH
regulations and ensure the safety, efficacy and shelf life of antibiotic formulations.

Keywords: RP-HPLC, Antibiotics, Method Development, Validation, Stability-Indicating Methods, Forced Degradation, ICH Q1A(R2), ICH Q2(R2), impurity profiling, QbD.