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.

Keywords: 4-epi-OTC; Huanglongbing; OTC; antibiotic; citrus; europium; oxytetracycline.

Figure 1

Complexation of europium with oxytetracycline (OTC). (A) Formation of the fluorescent 1:1 β-diketone:Eu(III) complex via the BCD (second to fourth) rings hydroxyl groups of OTC. The OTC absorbs. The OTC attached to the europium ion strongly absorbs at 388 nm and passes the absorbed energy to europium, which emits it as a narrow and intense peak at 615 nm. (B) OTC standard with and without europium. (C) Relative fluorescence intensity of OTC with and without europium. Values with p-values < 0.05 are significantly different using two-tailed student t-test. Assay was conducted using 100 µL standard, 300 µL Tris buffer (0.1 M, pH 8.5), 40 µL ethylenediaminetetraacetic acid (EDTA) (0.00025 M), 40 µL cetyltrimethylammonium chloride (CTAC), and with or without 20 µL europium (0.00125 M).

Figure 2

Interference of phenols and flavonoids with the europium method. (A) Relative fluorescence inhibition (%) of gallic acid and catechin when present in the final assay at 100 and 1000 ppm. (B) The UV-visible spectra of gallic acid and catechin with CTAC in tris buffer (pH 8.5). (C) Reaction scheme between CTAC and phenol. Values with p-values < 0.05 are significantly different using two-tailed Student t-test.

Figure 3

Effect of surfactant on the fluorescence intensity of the OTC-Eu(III) complex. (A) Fluorescence intensity of OTC standard with 1% CTCA, 4.5% CTCA, 10% Triton X-100, and without any surfactant. (B) Fluorescence intensity of OTC standard with 10% Triton X-100 and without any surfactant. (C) Fluorescence intensity of OTC standard (prepared in sample matrix and cleaned using 60 mg hydrophilic–lipophilic balanced (HLB) cartridge) with 1 and 4.5% CTCA. Assay was conducted using 100 µL standard, 300 µL Tris buffer (0.1 M, pH 8.5), 40 µL EDTA (0.00025 M), 20 µL europium (0.00125 M), and 40 µL surfactant.

Figure 4

Recovery of OTC from spiked citrus leaf samples using 60 mg HLB cartridge. (A) Standard curves of OTC prepared in sample matrix (control samples extracted using 1 M HCl containing 2.2% trichloroacetic acid with and without 0.1% EDTA) and cleaned by eluting 0.25 mL into the 60 mg HLB cartridge. (B) Percentage recoveries of OTC from spiked leaf samples extracted using 1 M HCl containing 2.2% trichloroacetic acid with and without 0.1% EDTA. Values with p-values < 0.05 are significantly different using two-tailed student t-test.

Figure 5

Application of europium method to filed samples and comparison with ELISA kit. OTC was extracted using the HCl and trichloroacetic acid mixture and cleaned using the HLB cartridge before being analyzed using the europium assay. The acidic extract was diluted (1:100) using distilled water, and then (1:4) using the dilution buffer provided by the kit and analyzed directly without any cleanup. Values with p-values < 0.05 are significantly different using the two-tailed student t-test.

Figure 6

Cross-reactivity of the OTC metabolites with the europium method. (A) Chemical structure of OTC metabolites; 4-epi-OTC, α-apo-OT, β-apo-OTC. (B) Standard curves 4-epi-OTC, α-apo-OTC, and β-apo-OTC, and OTC as generated using the europium method. (C) Cross-reactivity (%) of 4-epi-OTC, α-apo-OTC, and β-apo-OTC relative to that of OTC. Values with p-values < 0.05 are significantly different using two-tailed student t-test. Standards (5.0, 2.5, 1.2, 0.6, and 0.3 ppm) were dissolved in water. Assays were performed under the same conditions; 100 µL of standard, 300 µL of tris buffer, 40 µL of EDTA, 40 µL of 1% CTAC, and 20 µL of europium. Responses (relative fluorescence), at the same concentration, with different letters are significantly different using Tukey’s honestly significant difference (HSD) test (p-value < 0.05).

Figure 7

Extraction, cleanup, and europium assay for OTC extracted from citrus leaves. (A) Extraction scheme for OTC from ground citrus leaf tissues. (B) Cleanup of OTC from citrus leaf samples (0.25 mL) using 60 mg HLB cartridge. (C) Fluorescence assay of OTC.