Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues
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 develop...
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| Format: | Article |
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MDPI AG
2021-02-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/10/2/224 |
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| author | Faraj Hijaz Yasser Nehela Pedro Gonzalez-Blanco Nabil Killiny |
| author_facet | Faraj Hijaz Yasser Nehela Pedro Gonzalez-Blanco Nabil Killiny |
| author_sort | Faraj Hijaz |
| collection | DOAJ |
| description | 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<sup>−1</sup> 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-<i>epi</i>-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-<i>apo</i>-OTC and β-<i>apo</i>-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. |
| first_indexed | 2024-03-09T00:36:18Z |
| format | Article |
| id | doaj.art-3c264f4c25124a168d12ecaed3dfc5f3 |
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| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-09T00:36:18Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-3c264f4c25124a168d12ecaed3dfc5f32023-12-11T18:07:23ZengMDPI AGAntibiotics2079-63822021-02-0110222410.3390/antibiotics10020224Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus TissuesFaraj Hijaz0Yasser Nehela1Pedro Gonzalez-Blanco2Nabil Killiny3Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USADepartment of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USADepartment of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USADepartment of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USAAntimicrobial 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<sup>−1</sup> 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-<i>epi</i>-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-<i>apo</i>-OTC and β-<i>apo</i>-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.https://www.mdpi.com/2079-6382/10/2/224HuanglongbingoxytetracyclineOTCeuropium4-epi-OTCantibiotic |
| spellingShingle | Faraj Hijaz Yasser Nehela Pedro Gonzalez-Blanco Nabil Killiny Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues Antibiotics Huanglongbing oxytetracycline OTC europium 4-epi-OTC antibiotic |
| title | Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues |
| title_full | Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues |
| title_fullStr | Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues |
| title_full_unstemmed | Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues |
| title_short | Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues |
| title_sort | development of europium sensitized fluorescence based method for sensitive detection of oxytetracycline in citrus tissues |
| topic | Huanglongbing oxytetracycline OTC europium 4-epi-OTC antibiotic |
| url | https://www.mdpi.com/2079-6382/10/2/224 |
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