A sensitive HPLC method for quantitative determination of oxytetracycline and its structural analogues to support residue monitoring in disease-managed crops

Authors: YASSER, NEHALA - University Of Florida; VINCENT, CHRISTOPHER - University Of Florida; Heck, Michelle; KILLINY, NABIL - University Of Florida

Submitted to: Environmental Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2026
Publication Date: 1/11/2026
Citation: Yasser, N., Vincent, C., Heck, M.L., Killiny, N. 2026. A sensitive HPLC method for quantitative determination of oxytetracycline and its structural analogues to support residue monitoring in disease-managed crops. Environmental Research. 293. Article 123755. https://doi.org/10.1016/j.envres.2026.123755.
DOI: https://doi.org/10.1016/j.envres.2026.123755

Interpretive Summary: The use of oxytetracycline (OTC) to manage bacterial plant diseases, including citrus greening, has raised important questions about antibiotic residues, environmental safety, and regulatory compliance. To address these concerns, this study developed and validated a sensitive and reliable analytical method to accurately measure OTC and closely related compounds in citrus tissues. The method (high-performance liquid chromatography) was capable of separating and quantifying OTC and six structurally related analogues across multiple citrus matrices, including leaves, peel, pulp, and juice. The method demonstrated strong accuracy, reproducibility, and sensitivity, with detection limits well below current regulatory thresholds for citrus products. Importantly, the method performed consistently across healthy and disease-affected tissues, confirming its robustness under real-world conditions. When applied to samples collected from commercial citrus groves where OTC was administered via trunk injection the method detected OTC residues in leaf tissue but found no detectable residues in juice. No structurally related OTC analogues were detected in any edible citrus tissues. These findings indicate limited or no movement of OTC into consumable fruit and support current residue safety assessments for its use in commercial citrus production. Overall, this work provides a validated analytical tool that strengthens the scientific basis for monitoring antibiotic residues in citrus production and supports regulatory oversight, food safety assurance, and informed decision-making regarding disease management strategies in citrus groves.

Technical Abstract: Integrating oxytetracycline (OTC) and its structural analogues in management programs for bacterial plant diseases, such as citrus greening, remains controversial due to concerns over antimicrobial resistance, environmental impact, and residue safety, highlighting the urgent need for sensitive and reliable method(s) to monitor these compounds across complex plant tissue matrices. The current study introduces an HPLC-PDA-based method that was developed and validated for the quantitative determination of OTC and six structurally related analogues in citrus tissue matrices. Chromatographic separation was optimized using a gradient mobile phase combining 0.01 M oxalic acid (pH 2.5) and acetonitrile, which provided sharp, symmetrical peaks and baseline resolution of all OTC analogues, with low tailing factors (<1.2). Despite the overall OTC class similarity, UV spectral profiling confirmed the distinct spectral features of OTC analogues, supporting their identification, particularly where minor co-elution occurred. OTC calibration curves showed strong linearity across the tested range (0.195 - 100 µg.mL-1) with high significant fits (p <0.0001) and strong coefficients of determination (R2>0.99, except for TC and a-apo-OTC where R² >0.98), while intra- and inter-day precision remained within acceptable limits (RSDs <5%), confirming reliability and reproducibility of the developed method. Spiking studies exhibited reliable inter-matrix performance for the method with high extraction recovery (84-108%) and minimal matrix effects across citrus leaves (healthy and infected), peel, pulp, and juice. The developed method showed high sensitivity across different citrus tissues, with limits of detection (LOD) as low as 1.48 ng·mL'¹ and limits of quantification (LOQ) below the default regulatory threshold for citrus (10 parts per billion) set by the U.S. Environmental Protection Agency (EPA). Application to real-world leaf and juice samples collected from OTC-injected commercial groves revealed the presence of OTC parent compound residues, but not other OTC analogues, in leaves of injected trees, but no detectable translocation into juice. Taken together, the method offers a sensitive, reliable, and efficient platform for monitoring OTC residues in citrus tissues, supporting residue compliance and food safety in disease-managed production systems.