Abstract

Integrating oxytetracycline (OTC) in management programs of bacterial plant diseases, including citrus greening, remains controversial due to concerns related to antimicrobial resistance, environmental impact, and residue safety, highlighting the need for sensitive and reliable residue monitoring methods. This study reports the development and validation of an HPLC-PDA-based method for the quantitative determination of OTC and six structurally related analogues in citrus tissue. Chromatographic separation was achieved using a gradient mobile phase of 0.01 M oxalic acid (pH 2.5) and acetonitrile, providing baseline resolution, symmetrical peak shapes, and low tailing factors (<1.2) for all target analytes. Despite structural similarity among OTC analogues, UV spectral profiling enabled reliable compound discrimination, even in cases of minor co-elution. The method demonstrated strong linearity over a concentration range of 0.195-100 μg mL-1 (R2 >0.99 for most analytes), with acceptable intra- and inter-day precision (RSD <5%). Spiking studies exhibited reliable inter-matrix performance with extraction recoveries ranging from 84 to 108% across multiple citrus matrices, including healthy and infected leaves, peel, pulp, and juice, with minimal matrix interference. The method showed high sensitivity across citrus tissues, with limits of detection (LOD) as low as 1.48 ng mL-1 and limits of quantification (LOQ) below the U.S. Environmental Protection Agency (EPA) default regulatory threshold for citrus. Application to field samples from OTC-injected commercial groves detected parent OTC residues in citrus leaves, but not other structural analogues, and no detectable OTC translocation into juice. Overall, this method provides a robust analytical platform that enables simultaneous, high-sensitivity quantification of OTC and multiple structural analogues across complex citrus matrices to support environmental exposure assessment, regulatory compliance, and food safety evaluation in disease-managed citrus production systems.