Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues

Abstract

Antimicrobial compounds have been successfully used to control many plant and animal diseases. Recently, oxytetracycline (OTC) and streptomycin have been approved for the treatment of Huanglongbing in citrus. Since the application of OTC is under strict regulations, several methods have been developed to determine and monitor its levels in the environment including high-performance liquid chromatography, ELISA, colorimetric, and fluorometric assays. In this study, we developed a fluorometric method for the determination of OTC in plant tissues based on its complexation with europium. Our preliminary trials showed that phenols and flavonoids interfere with the europium assay by reacting with the sensitizing reagent, cetyltrimethylammonium chloride. Consequently, we used the 60 mg hydrophilic–lipophilic balanced (HLB) cartridges to purify the OTC from the plant matrix. The recovery of OTC from spiked leaf samples was 75 ± 7.6%. Using the 500 mg HLB, we were able to detect 0.3 ppm OTC in the final sample extract, which corresponds to 3 µg g−1 fresh weight (FWT). The developed method was successfully used to measure the level of OTC in leaves obtained from trunk-injected trees. The results obtained by the europium method were similar to those obtained using the ELISA assay. We also tested the cross-reactivity of OTC metabolites with the europium method. The 4-epi-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-apo-OTC and β-apo-OTC showed small cross-reactivity. We showed that the europium-sensitized fluorescence-based method can be successfully used to assess OTC in citrus plant tissues after a cleanup step. Our results showed that this method was sensitive, reproducible, and can be used to analyze many samples simultaneously.

Citrate Mediated Europium-Based Detection of Oxytetracycline in Citrus Tissues

“…Although many methods have been established for the detection of OTC including liquid chromatography and several colorimetric methods, the enzyme-linked immunosorbent assay (ELISA) is considered the most preferred method [ 10 ]. The ELISA method is fast, simple, sensitive, and can be used to examine many samples at the same time [ 10 ].…”

“…Although many methods have been established for the detection of OTC including liquid chromatography and several colorimetric methods, the enzyme-linked immunosorbent assay (ELISA) is considered the most preferred method [ 10 ]. The ELISA method is fast, simple, sensitive, and can be used to examine many samples at the same time [ 10 ]. In our previous studies, we used the ELISA kit to study the uptake and translocation of OTC in citrus plants after root drench, trunk injection, and foliar application [ 7 , 10 ].…”

“…The ELISA method is fast, simple, sensitive, and can be used to examine many samples at the same time [ 10 ]. In our previous studies, we used the ELISA kit to study the uptake and translocation of OTC in citrus plants after root drench, trunk injection, and foliar application [ 7 , 10 ]. Although the OTC ELISA kit is fast and sensitive, it is expensive and is not available in the market all the time.…”

“…The europium method has been successfully used to detect several tetracyclines in different matrixes including meat, milk, serum, and urine [ 12 , 13 ]. Although the europium method is sensitive and has been known for more than twenty-five years, it has not been applied to plant tissues until 2021 [ 10 ].…”

“…In our previous study, we established a fluorometric method for the detection of OTC in citrus leaves by complexing it with europium and cetyltrimethylammonium chloride (CTAC) [ 10 ]. We also found that phenols could interfere with europium assay by reacting with the sensitizing reagent, CTAC [ 10 ]. To avoid this interference, we replaced the CTCA with Triton X-100.…”