Comprehensive evaluation of analytical methods for determination of ansamycins across diverse matrices

Ansamycins (ANs), including rifampicin (RIF), rifabutin (RFB), rifapentine (RPT), rifalazil (RFL), and rifaximin (RFX), are structurally distinctive macrocyclic antibiotics whose analytical determination is complicated by very low systemic exposure (especially RFX), high protein binding and intracellular sequestration (notably RFB and RFL), and extensive hepatic metabolism. The reviewed articles were retrieved from major scientific databases, including Scopus, Web of Science, ScienceDirect, Google Scholar, and PubMed, and were limited to English-language publications. This review aims to provide a comprehensive and comparative evaluation of chromatographic, spectroscopic, and electroanalytical strategies used over the past 25 years to quantify clinically important ANs across biological and pharmaceutical matrices, linking analytical performance to drug-specific physicochemical properties, stability constraints, and bioanalytical applications. The reviewed literature shows that high-performance liquid chromatography remains the core methodology, with LC-MS/MS representing the gold standard for trace-level determination, particularly for RFB, RPT, RFL, and RFX, due to its superior structural selectivity and sensitivity, whereas UV-based HPLC remains suitable mainly for pharmaceutical quality control because co-absorbing rifamycin derivatives frequently compromise accuracy in biological matrices, while electroanalytical and spectroscopic methods, despite their simplicity and lower cost, face limitations in complex matrices due to selectivity constraints. Overall, matrix complexity and analyte instability were the dominant contributors to variability in AN quantification. Looking forward, advances are expected to arise from continued refinement of LC-MS/MS workflows, greater use of orthogonal separation strategies, improved stabilization protocols, and sustainable, miniaturized sample-preparation approaches, thereby ensuring reliable, high-sensitivity AN determination to support therapeutic drug monitoring, clinical pharmacokinetics, and pharmaceutical quality control.